Malingaliro osiyanasiyana okhudza chitetezo cha zomera amapereka malangizo ofunikira pofotokoza momwe zomera zimagwirira ntchito, koma maulosi awo ofunikira akadali kuyesedwa. Pano, tagwiritsa ntchito kusanthula kwa tandem mass spectrometry (MS/MS) kopanda tsankho kuti tifufuze bwino metabolome ya mitundu ya fodya yochepetsedwa kuchokera ku zomera zosiyanasiyana kupita ku mitundu ina ndi mitundu yogwirizana, ndipo tagwiritsa ntchito malingaliro ambiri okhudza mawonekedwe a mass spectrometric kutengera ma compound spectra mu chidziwitsocho. Chikhazikitso choyesa maulosi ofunikira a optimal defense (OD) ndi moving target (MT) theories. Gawo la chidziwitso cha metabolomics cha zomera likugwirizana ndi chiphunzitso cha OD, koma limatsutsana ndi maulosi ofunikira a chiphunzitso cha MT pa metabolomics dynamics yoyambitsidwa ndi herbivores. Kuyambira pa micro mpaka macro evolutionary scale, chizindikiro cha jasmonate chinadziwika ngati chizindikiro chachikulu cha OD, pomwe chizindikiro cha ethylene chinapereka kusintha kwa mayankho enieni a herbivore omwe afotokozedwa ndi netiweki ya MS/MS molecular.
Ma metabolites apadera okhala ndi kapangidwe kosiyanasiyana ndi omwe amatenga nawo mbali kwambiri pakusinthasintha kwa zomera ku chilengedwe, makamaka poteteza adani (1). Kusiyanasiyana kodabwitsa kwa kagayidwe kachakudya kapadera komwe kamapezeka m'zomera kwalimbikitsa kafukufuku wozama wazaka zambiri pazinthu zambiri za chilengedwe, ndipo kwapanga mndandanda wautali wa malingaliro oteteza zomera, omwe ndi chitukuko cha kusintha kwa chilengedwe ndi chilengedwe cha kuyanjana kwa zomera ndi tizilombo. Kafukufuku wowona bwino amapereka chitsogozo chofunikira (2). Komabe, malingaliro oteteza zomera awa sanatsatire njira yokhazikika ya kulingalira koganizira, momwe maulosi ofunikira anali pamlingo womwewo wa kusanthula (3) ndipo adayesedwa mwa kuyesa kuti apititse patsogolo kuzungulira kwotsatira kwa chitukuko cha chiphunzitso (4). Zoletsa zaukadaulo zimaletsa kusonkhanitsa deta kumagulu enaake a kagayidwe kachakudya ndikuchotsa kusanthula kwathunthu kwa ma metabolites apadera, motero kuletsa kufananiza kwa magulu osiyanasiyana komwe ndikofunikira pakukula kwa chiphunzitso (5). Kusowa kwa deta yonse ya metabolomics ndi ndalama yofanana yoyerekeza kayendedwe ka ntchito ka kagayidwe kachakudya pakati pa magulu osiyanasiyana a zomera kumalepheretsa kukula kwasayansi kwa munda.
Zochitika zaposachedwa kwambiri m'munda wa tandem mass spectrometry (MS/MS) metabolomics zitha kufotokoza bwino kusintha kwa kagayidwe kachakudya mkati ndi pakati pa mitundu ya dongosolo linalake, ndipo zitha kuphatikizidwa ndi njira zowerengera kuti ziwerengere kufanana kwa kapangidwe kake pakati pa zosakaniza zovutazi. Chidziwitso choyambirira cha chemistry (5). Kuphatikiza kwa ukadaulo wapamwamba mu kusanthula ndi kugwiritsa ntchito makompyuta kumapereka maziko ofunikira pakuyesa kwanthawi yayitali kwa maulosi ambiri opangidwa ndi malingaliro achilengedwe ndi kusintha kwa kusiyanasiyana kwa kagayidwe kachakudya. Shannon (6) adayambitsa chiphunzitso cha chidziwitso koyamba m'nkhani yake yayikulu mu 1948, ndikuyika maziko a kusanthula kwa masamu kwa chidziwitso, komwe kwagwiritsidwa ntchito m'magawo ambiri kupatula kugwiritsa ntchito koyambirira. Mu genomics, chiphunzitso cha chidziwitso chagwiritsidwa ntchito bwino kuti awerengere chidziwitso chosunga nthawi (7). Mu kafukufuku wa transcriptomics, chiphunzitso cha chidziwitso chimasanthula kusintha konse mu transcriptome (8). Mu kafukufuku wakale, tidagwiritsa ntchito dongosolo la ziwerengero la chiphunzitso cha chidziwitso ku metabolomics kuti tifotokoze ukatswiri wa kagayidwe kachakudya wa mulingo wa minofu muzomera (9). Apa, tikuphatikiza njira yogwirira ntchito yochokera ku MS/MS ndi dongosolo la ziwerengero la chiphunzitso cha chidziwitso, chomwe chimadziwika ndi kusiyanasiyana kwa kagayidwe kachakudya mu ndalama yofanana, kuti tiyerekezere maulosi ofunikira a chiphunzitso cha chitetezo cha zomera cha metabolome yoyambitsidwa ndi nyama zodya udzu.
Mapangidwe a chiphunzitso cha chitetezo cha zomera nthawi zambiri amakhala ogwirizana ndipo amatha kugawidwa m'magulu awiri: omwe amayesa kufotokoza kufalikira kwa ma metabolites enieni a zomera kutengera ntchito zodzitetezera, monga chitetezo chabwino kwambiri (OD) (10), cholinga chosuntha (MT) (11)) Ndi chiphunzitso cha maonekedwe (12), pomwe ena amafuna kufotokozera kwamakina momwe kusintha kwa kupezeka kwa zinthu kumakhudzira kukula kwa zomera ndi kusonkhanitsa ma metabolites apadera, monga kaboni: lingaliro la nutrient balance (13), lingaliro la kukula kwa chiŵerengero cha kukula (14), ndi lingaliro la kukula ndi kusiyana kwa equilibrium (15). Magulu awiri a malingaliro ali pamlingo wosiyana wa kusanthula (4). Komabe, malingaliro awiri okhudzana ndi ntchito zodzitetezera pamlingo wogwirira ntchito amalamulira zokambirana za chitetezo chokhazikika cha zomera ndi chokhazikika: lingaliro la OD, lomwe likuganiza kuti zomera zimayika ndalama mu chitetezo chawo chamankhwala chokwera mtengo pokhapokha ngati pakufunika, mwachitsanzo, zikamezedwa Pamene nyama ya udzu ikuukira, motero, malinga ndi kuthekera kwa kuukira mtsogolo, chinthu chokhala ndi ntchito yodzitetezera chimaperekedwa (10); Lingaliro la MT likusonyeza kuti palibe kusintha kwa metabolite komwe kumachitika mbali imodzi, koma metabolite imasintha mwachisawawa, motero imapanga kuthekera koletsa "cholinga choyenda" cha kagayidwe kachakudya cha nyama zodya udzu. Mwanjira ina, malingaliro awiriwa amapereka maulosi osiyana okhudza kusintha kwa kagayidwe kachakudya komwe kumachitika pambuyo pa kuukira kwa nyama zodya udzu: ubale pakati pa kusonkhanitsa kwa metabolite mbali imodzi ndi ntchito yoteteza (OD) ndi kusintha kwa kagayidwe kachakudya kosalunjika (MT) (11).
Malingaliro a OD ndi MT samangokhudza kusintha komwe kumachitika mu metabolome, komanso zotsatira za chilengedwe ndi kusintha kwa zinthu zomwe zimasonkhanitsidwa, monga ndalama zosinthira ndi ubwino wa kusintha kwa kagayidwe kachakudya m'malo enaake achilengedwe (16). Ngakhale malingaliro onsewa amazindikira ntchito yoteteza ya ma metabolites apadera, omwe angakhale okwera mtengo kapena osakwera mtengo, kulosera kwakukulu komwe kumasiyanitsa malingaliro a OD ndi MT kuli mu momwe kusintha kwa kagayidwe kachakudya komwe kumachitika kumayambira. Kulosera kwa chiphunzitso cha OD kwalandira chidwi chachikulu kwambiri mpaka pano. Mayesowa akuphatikizapo kuphunzira za ntchito zoteteza mwachindunji kapena zosalunjika za minofu yosiyanasiyana ya mankhwala enaake m'malo obiriwira ndi m'malo achilengedwe, komanso kusintha kwa gawo la kukula kwa zomera (17-19). Komabe, mpaka pano, chifukwa cha kusowa kwa njira yogwirira ntchito ndi njira zowerengera za kusanthula kwathunthu kwa kusiyanasiyana kwa kagayidwe kachakudya padziko lonse lapansi, kusiyana kwakukulu pakati pa malingaliro awiriwa (ndiko kuti, njira yosinthira kagayidwe kachakudya) kukadali koyenera kuyesedwa. Pano, tikupereka kusanthula kotere.
Chimodzi mwa zizindikiro zofunika kwambiri za metabolites za zomera ndi kusiyanasiyana kwawo kwakukulu pamagulu onse kuyambira chomera chimodzi, kuchuluka kwa anthu mpaka mitundu yofanana (20). Kusintha kwakukulu kwa kuchuluka kwa metabolites zapadera kumatha kuwoneka pa kuchuluka kwa anthu, pomwe kusiyana kwakukulu kwabwino nthawi zambiri kumakhalabe pamlingo wa mitundu (20). Chifukwa chake, kusiyanasiyana kwa kagayidwe ka zomera ndiye gawo lalikulu la kusiyanasiyana kwa ntchito, kuwonetsa kusinthasintha kwa malo osiyanasiyana, makamaka malo omwe ali ndi kuthekera kosiyana kwa kuukira ndi tizilombo tapadera ndi zomera wamba (21). Kuyambira nkhani ya Fraenkel (22) yokhudza zifukwa zomwe zilili ndi metabolites za zomera, kuyanjana ndi tizilombo tosiyanasiyana kwawonedwa ngati zovuta zofunika kusankha, ndipo kuyanjana kumeneku kumakhulupirira kuti kunapanga zomera panthawi ya kusintha. Njira ya kagayidwe ka zinthu (23). Kusiyana kwa mitundu yosiyanasiyana ya metabolites zapadera kungasonyezenso kulinganiza kwa thupi komwe kumagwirizanitsidwa ndi chitetezo cha zomera zomwe zimapanga komanso zomwe zingayambitse njira zowononga zomera, chifukwa mitundu iwiriyi nthawi zambiri imagwirizana molakwika (24). Ngakhale zingakhale zothandiza kukhala ndi chitetezo chabwino nthawi zonse, kusintha kwa kagayidwe kachakudya komwe kumachitika panthawi yake komwe kumakhudzana ndi chitetezo kumapereka ubwino womveka bwino polola zomera kugawa zinthu zofunika kuzinthu zina za thupi (19, 24), ndikupewa kufunikira kwa mgwirizano. Kuwonongeka kwa ndalama (25). Kuphatikiza apo, kukonzanso kumeneku kwa metabolites apadera omwe amayambitsidwa ndi tizilombo todya zomera kungayambitse kufalikira kowononga mwa anthu (26), ndipo kungawonetse kuwerengedwa mwachindunji kwa kusintha kwakukulu kwachilengedwe mu chizindikiro cha jasmonic acid (JA), chomwe chingasungidwe mwa anthu. Zizindikiro zapamwamba ndi zochepa za JA ndi malonda pakati pa chitetezo motsutsana ndi zomera zodya zomera ndi mpikisano ndi mitundu inayake (27). Kuphatikiza apo, njira zapadera za metabolite biosynthetic zidzawonongeka mwachangu komanso kusinthika panthawi ya kusintha, zomwe zimapangitsa kuti kagayidwe kachakudya ...
Apa, tathetsa mavuto awa mwachindunji. (I) Kodi tizilombo todya zomera timasintha bwanji metabolome ya chomera? (Ii) Kodi ndi zigawo ziti zazikulu za chidziwitso cha metabolic plasticity zomwe zingayesedwe kuti ziyese maulosi a chiphunzitso cha chitetezo cha nthawi yayitali? (Iii) Kodi kusintha metabolome ya chomera mwanjira yapadera kwa wowukira, ngati ndi choncho, kodi mahomoni a chomera amachita gawo lotani pakusintha yankho la kagayidwe kachakudya, ndipo ndi ma metabolites ati omwe amathandizira ku mtundu wa chitetezo? (Iv) Popeza maulosi opangidwa ndi malingaliro ambiri a chitetezo amatha kufalikira m'magawo onse a minofu ya zamoyo, tinafunsa kuti yankho la kagayidwe kachakudya lomwe limayambitsidwa ndi lofanana bwanji kuyambira kufananiza mkati mpaka kufananiza mitundu yosiyanasiyana? Pachifukwa ichi, taphunzira mwadongosolo metabolome ya masamba a fodya wa nikotini, womwe ndi chomera chachilengedwe chokhala ndi kagayidwe kapadera, ndipo umagwira ntchito motsutsana ndi mphutsi za zomera ziwiri zakuthengo, Lepidoptera Datura (Ms) ( Wamphamvu kwambiri, makamaka wodyedwa) Pa Solanaceae ndi Spodoptera littoralis (Sl), nyongolotsi za masamba a thonje ndi mtundu wa "mtundu", ndi zomera za Solanaceae zomwe zimakhalapo ndi mitundu ina ya mbewu ndi mabanja. Tinafufuza za MS/MS metabolomics spectrum ndikupeza mafotokozedwe a ziwerengero za chidziwitso kuti tiyerekezere malingaliro a OD ndi MT. Pangani mamapu apadera kuti awulule kudziwika kwa metabolites ofunikira. Kusanthulaku kunakulitsidwa kwa anthu obadwira ku N. nasi ndi mitundu ya fodya yogwirizana kwambiri kuti tiwunikenso bwino covariance pakati pa chizindikiro cha mahomoni a zomera ndi OD induction.
Kuti tijambule mapu onse okhudza kupangika kwa metabolome ya masamba a fodya wodya zomera, tinagwiritsa ntchito njira yowunikira komanso kuwerengera yomwe idapangidwa kale kuti tisonkhanitse mokwanira ndikuchotsa deta ya MS/MS spectra yodziyimira payokha kuchokera ku zotulutsa za zomera (9). Njira iyi yosasinthika (yotchedwa MS/MS) imatha kupanga ma spectra osagwiritsidwa ntchito kwambiri, omwe angagwiritsidwe ntchito pa kusanthula konse kwa magawo omwe afotokozedwa pano. Ma metabolites a zomera osakhudzidwa awa ndi amitundu yosiyanasiyana, okhala ndi mazana mpaka zikwi za metabolites (pafupifupi 500-1000-s/MS/MS pano). Pano, tikuganizira za kupangika kwa metabolome mu dongosolo la chidziwitso, ndikuwerengera kusiyanasiyana ndi ukatswiri wa metabolome kutengera Shannon entropy ya kugawa kwa frequency ya metabolic. Pogwiritsa ntchito njira yomwe idagwiritsidwa ntchito kale (8), tinawerengera zizindikiro zomwe zingagwiritsidwe ntchito kuyeza kusiyanasiyana kwa metabolome (Hj indicator), specialization ya metabolic profile (δj indicator) ndi metabolic specificity ya metabolite imodzi (Si indicator). Kuphatikiza apo, tinagwiritsa ntchito Relative Distance Plasticity Index (RDPI) kuti tiyese kuchuluka kwa metabolome inducibility ya herbivores (Chithunzi 1A) (30). Mu dongosolo la ziwerengerozi, timagwiritsa ntchito MS/MS spectrum ngati gawo loyambira la chidziwitso, ndikusintha kuchuluka kwa MS/MS kukhala mapu ogawa pafupipafupi, kenako timagwiritsa ntchito Shannon entropy kuti tiyerekeze kusiyana kwa metabolome kuchokera pamenepo. Kusankha kwa metabolome kumayesedwa ndi avareji yeniyeni ya MS/MS spectrum imodzi. Chifukwa chake, kuwonjezeka kwa kuchuluka kwa magulu ena a MS/MS pambuyo pa kulowetsedwa kwa herbivore kumasinthidwa kukhala spectral inducibility, RDPI ndi specialization, ndiko kuti, kuwonjezeka kwa δj index, chifukwa metabolites zapadera kwambiri zimapangidwa ndipo high Si index imapangidwa. Kutsika kwa Hj diversity index kukuwonetsa kuti chiwerengero cha MS/MS yopangidwa chachepetsedwa, kapena kugawa kwa profile frequency kumasintha pang'ono, pomwe kuchepetsa kusatsimikizika kwake konse. Kudzera mu kuwerengera kwa Si index, n'zotheka kuwonetsa kuti ndi MS/MS iti yomwe imayambitsidwa ndi nyama zina zodya udzu, m'malo mwake, ndi MS/MS iti yomwe simayankha ku induction, yomwe ndi chizindikiro chofunikira chosiyanitsa kulosera kwa MT ndi OD.
(A) Zofotokozera ziwerengero zomwe zimagwiritsidwa ntchito pa zomera (H1 mpaka Hx) MS/MS data-inducibility (RDPI), kusiyanasiyana (Hj index), specialization (δj index) ndi metabolite specificity (Si index). Kuwonjezeka kwa digiri ya specialization (δj) kumasonyeza kuti, pa avareji, metabolites zambiri za zomera zidzapangidwa, pomwe kuchepa kwa kusiyanasiyana (Hj) kumasonyeza kuchepa kwa kupanga metabolites kapena kugawa kosagwirizana kwa metabolites mu mapu ogawa. Mtengo wa Si umayesa ngati metabolite ndi yeniyeni pa mkhalidwe winawake (pano, herbivorous) kapena mosiyana ikusungidwa pamlingo womwewo. (B) Chithunzi cha lingaliro la kuneneratu kwa chiphunzitso cha chitetezo pogwiritsa ntchito mfundo za chidziwitso. Chiphunzitso cha OD chimaneneratu kuti kuukira kwa herbivore kudzawonjezera metabolites za chitetezo, motero kumawonjezera δj. Nthawi yomweyo, Hj imachepa chifukwa mbiriyo imakonzedwanso kuti ichepetse kusatsimikizika kwa chidziwitso cha kagayidwe kachakudya. Chiphunzitso cha MT chimaneneratu kuti kuukira kwa herbivores kudzayambitsa kusintha kosalunjika mu metabolome, motero kumawonjezera Hj ngati chizindikiro cha kuchuluka kwa kusatsimikizika kwa chidziwitso cha kagayidwe kachakudya ndikuyambitsa kufalikira kwa Si mwachisawawa. Tinaperekanso chitsanzo chosakanikirana, MT yabwino kwambiri, momwe ma metabolites ena okhala ndi chitetezo champhamvu adzawonjezeka kwambiri (mtengo wapamwamba wa Si), pomwe ena amawonetsa mayankho mwachisawawa (mtengo wotsika wa Si).
Pogwiritsa ntchito mafotokozedwe a chiphunzitso cha chidziwitso, timatanthauzira chiphunzitso cha OD kuti tinene kuti kusintha kwapadera kwa metabolite komwe kumachitika chifukwa cha herbivore mu mkhalidwe wosapangidwa kudzatsogolera ku (i) kuwonjezeka kwa metabolic specificity (Si index) kuyendetsa metabonomic specificity (δj index) Kuwonjezeka kwa) magulu ena apadera a metabolite omwe ali ndi chitetezo chambiri, ndi (ii) kuchepa kwa metabolome diversity (Hj index) chifukwa cha kusintha kwa metabolic frequency distribution kupita ku leptin body distribution yambiri. Pa mulingo wa metabolite imodzi, kugawa kwa Si kolamulidwa kumayembekezeredwa, komwe metabolite idzawonjezera Si value malinga ndi chitetezo chake (Chithunzi 1B). Motsatira izi, tikufotokozera chiphunzitso cha MT kuti tinene kuti kusangalatsa kudzatsogolera ku (i) kusintha kosalunjika kwa metabolites komwe kumabweretsa kuchepa kwa δj index, ndi (ii) kuwonjezeka kwa Hj index chifukwa cha kuwonjezeka kwa metabolic infidelity. Kapena randomness, yomwe ingayesedwe ndi Shannon entropy mu mawonekedwe a generalized diversity. Ponena za kapangidwe ka metabolic, chiphunzitso cha MT chidzaneneratu kugawa kwachisawawa kwa Si. Poganizira kuti ma metabolites ena ali pansi pa mikhalidwe inayake, ndipo mikhalidwe ina siili pansi pa mikhalidwe yeniyeni, ndipo kufunika kwawo kwa chitetezo kumadalira chilengedwe, tinaperekanso chitsanzo chosakanikirana cha chitetezo, momwe δj ndi Hj zimagawidwa m'magulu awiri motsatira Si Increase mbali zonse, magulu ena a metabolite okha, omwe ali ndi chitetezo chapamwamba, ndi omwe adzawonjezera makamaka Si, pomwe ena adzakhala ndi kugawa mwachisawawa (Chithunzi 1B).
Pofuna kuyesa kuneneratu kwa chiphunzitso chodziteteza pa mzere wa chofotokozera mfundo za chidziwitso, tinapeza mphutsi za herbivore za akatswiri (Ms) kapena generalist (Sl) pamasamba a Nepenthes pallens (Chithunzi 2A). Pogwiritsa ntchito kusanthula kwa MS/MS, tinapeza ma spectra 599 a MS/MS osagwiritsidwa ntchito kwambiri (fayilo ya data S1) kuchokera ku methanol extracts ya minofu ya masamba yomwe inasonkhanitsidwa pambuyo podya mbozi. Pogwiritsa ntchito ma index a RDPI, Hj, ndi δj kuti tiwone kusinthidwa kwa zomwe zili mu mafayilo a MS/MS configuration kumavumbula mapangidwe osangalatsa (Chithunzi 2B). Chizolowezi chonse ndichakuti, monga momwe chofotokozera mfundo chimafotokozera, pamene mbozi zikupitiriza kudya masamba, kuchuluka kwa kukonzanso kagayidwe kachakudya kumawonjezeka pakapita nthawi: maola 72 pambuyo poti zomera zidya, RDPI imawonjezeka kwambiri. Poyerekeza ndi kulamulira kosawonongeka, Hj idachepetsedwa kwambiri, zomwe zidachitika chifukwa cha kuchuluka kwapadera kwa mawonekedwe a kagayidwe kachakudya, komwe kudayesedwa ndi index ya δj. Kachitidwe kameneka kakugwirizana ndi zomwe zanenedweratu za chiphunzitso cha OD, koma sikagwirizana ndi zomwe zanenedweratu za chiphunzitso cha MT, chomwe chimakhulupirira kuti kusintha kosasinthika (kosakhala mbali) kwa milingo ya metabolite kumagwiritsidwa ntchito ngati njira yodzitetezera (Chithunzi 1B). Ngakhale kuti kuchuluka kwa zotulutsa mkamwa (OS) ndi kachitidwe ka chakudya cha nyama ziwirizi ndizosiyana, kudya kwawo mwachindunji kunapangitsa kusintha kofanana m'njira za Hj ndi δj panthawi yokolola ya maola 24 ndi maola 72. Kusiyana kokhako kunachitika pa maola 72 a RDPI. Poyerekeza ndi zomwe zachitika chifukwa cha kudya kwa Ms, kagayidwe kachakudya komwe kamachitika chifukwa cha kudya kwa Sl kanali kokwera.
(A) Kapangidwe koyesera: nkhumba wamba (S1) kapena katswiri (Ms) amadyetsedwa masamba a zomera zouma zomwe zachotsedwa mchere, pomwe pa zomera zofananira, OS ya Ms (W + OSM) imagwiritsidwa ntchito kuthana ndi kubowola kwa bala la masamba. Mphutsi za S1 (W + OSSl) kapena madzi (W + W). Kulamulira (C) ndi tsamba losawonongeka. (B) Kusasinthika (RDPI poyerekeza ndi tchati chowongolera), kusiyanasiyana (Hj index) ndi specialization (δj index) index yowerengedwa pamapu apadera a metabolite (599 MS/MS; fayilo ya data S1). Asterisks akuwonetsa kusiyana kwakukulu pakati pa kudyetsa mwachindunji kwa zomera zouma ndi gulu lowongolera (kuyesa kwa t kwa wophunzira ndi mayeso a t ophatikizidwa, *P<0.05 ndi ***P<0.001). ns, sizofunikira. (C) Chizindikiro cha nthawi yosinthira ya chinthu chachikulu (bokosi labuluu, amino acid, organic acid ndi shuga; fayilo ya data S2) ndi spectrum yapadera ya metabolite (bokosi lofiira 443 MS/MS; fayilo ya data S1) pambuyo pa chithandizo choyerekeza cha herbivory. Gulu la mtundu limatanthauza nthawi yodalirika ya 95%. Asterisk imasonyeza kusiyana kwakukulu pakati pa chithandizo ndi kulamulira [quadratic analysis of variance (ANOVA), kutsatiridwa ndi kusiyana kwakukulu kwa Tukey (HSD) kwa kufananiza kwa post hoc, *P<0.05, **P<0.01 Ndi *** P <0.001]. (D) Kusankha bwino malo osweka ndi ma profiles apadera a metabolite (zitsanzo zobwerezabwereza ndi mankhwala osiyanasiyana).
Kuti tifufuze ngati kusintha kwa zomera zomwe zimayambitsa matenda a herbivore pamlingo wa metabolome kumawonekera pakusintha kwa kuchuluka kwa metabolites payekhapayekha, choyamba tidayang'ana kwambiri pa metabolites zomwe zidaphunziridwa kale m'masamba a Nepenthes pallens omwe ali ndi kukana kwa herbivore kotsimikizika. Phenolic amides ndi ma hydroxycinnamamide-polyamine conjugates omwe amasonkhana panthawi ya njira yogwiritsira ntchito zomera za tizilombo ndipo amadziwika kuti amachepetsa magwiridwe antchito a tizilombo (32). Tinafufuza zoyambira za MS/MS yofanana ndi iyi ndipo tinajambula ma cumulative kinetic curves awo (Chithunzi S1). Mosadabwitsa, zinthu zochokera ku phenol zomwe sizikhudzidwa mwachindunji ndi chitetezo cha zomera, monga chlorogenic acid (CGA) ndi rutin, zimachepetsedwa pambuyo pa zomera. Mosiyana ndi zimenezi, zomera zimatha kupanga phenol amides kukhala yamphamvu kwambiri. Kudyetsa kosalekeza kwa zomera ziwirizi kunapangitsa kuti phenolamides ikhale ndi mphamvu yofanana, ndipo izi zinali zoonekeratu makamaka pakupanga kwa devo novo kwa phenolamides. Chomwecho chidzawonekanso pofufuza njira ya 17-hydroxygeranyl nonanediol diterpene glycosides (17-HGL-DTGs), yomwe imapanga ma diterpenes ambiri acyclic okhala ndi ntchito zotsutsana ndi zomera (33), zomwe Ms Feeding with Sl zinayambitsa mawonekedwe ofanana (Chithunzi S1)).
Vuto lomwe lingakhalepo poyesa kudya zomera mwachindunji ndi kusiyana kwa kuchuluka kwa masamba omwe amadya komanso nthawi yomwe amadya zomera, zomwe zimapangitsa kuti zikhale zovuta kuchotsa zotsatira za zomera zomwe zimayambitsidwa ndi mabala ndi zomera zomwe zimadya zomera. Pofuna kuthetsa bwino momwe mitundu ya zomera imayankhira kagayidwe ka masamba, tinayesa kudyetsa mphutsi za Ms ndi Sl mwa kugwiritsa ntchito nthawi yomweyo OS (OSM ndi OSS1) yomwe yasonkhanitsidwa kumene ku W yomwe ili ndi masamba okhazikika. Njirayi imatchedwa chithandizo cha W + OS, ndipo imasintha momwe mbewuyo imayankhira mwa kuyika nthawi yeniyeni yoyambira kwa yankho lomwe limayambitsidwa ndi zomera popanda kuyambitsa zotsatira zosokoneza za kusiyana kwa kuchuluka kapena kuchuluka kwa kutayika kwa minofu (Chithunzi 2A) (34). Pogwiritsa ntchito njira yowunikira ndi kuwerengera ya MS/MS, tinapeza ma spectra 443 a MS/MS (fayilo ya data S1), yomwe idagwirizana ndi ma spectra omwe adasonkhanitsidwa kale kuchokera ku kuyesa kwa chakudya mwachindunji. Kusanthula kwa chidziwitso cha deta ya MS/MS iyi kunawonetsa kuti kukonzanso kwa metabolomes yodziwika bwino ndi masamba poyesa herbivores kunawonetsa zoyambitsa za OS (Chithunzi 2C). Makamaka, poyerekeza ndi chithandizo cha OSS1, OSM idapangitsa kuti metabolome specialization ikhale yowonjezereka pa maola 4. Ndikofunikira kudziwa kuti poyerekeza ndi deta yoyesera ya herbivore feeding, metabolic kinetics yomwe idawonedwa mu malo awiri pogwiritsa ntchito Hj ndi δj ngati ma coordinates ndi momwe metabolome specialization imayendera poyankha chithandizo cha herbivore chomwe chidayesedwa pakapita nthawi. Kuwonjezeka kokhazikika (Chithunzi 2D). Nthawi yomweyo, tinayesa kuchuluka kwa amino acid, organic acids ndi shuga (data file S2) kuti tifufuze ngati kuwonjezeka kumeneku kwa luso la metabolome kuli chifukwa cha kusintha kwa metabolism ya carbon central poyankha herbivores yomwe idayesedwa (Chithunzi S2). Kuti tifotokoze bwino chitsanzo ichi, tinayang'anitsitsa kwambiri metabolic accumulation kinetics ya njira zomwe zidakambidwa kale za phenolamide ndi 17-HGL-DTG. Kulowetsedwa kwa zomera zamtundu wa OS kumasinthidwa kukhala njira yosiyana yokonzanso mkati mwa kagayidwe ka phenolamide (Chithunzi S3). Ma phenolic amides okhala ndi coumarin ndi caffeoyl moieties amayambitsidwa makamaka ndi OSS1, pomwe ma OSM amayambitsa kulowetsedwa kwina kwa ferulyl conjugates. Pa njira ya 17-HGL-DTG, kulowetsedwa kwa OS kosiyana ndi zinthu zotsika ndi malonylation ndi dimalonylation kunapezeka (Chithunzi S3).
Kenako, tinaphunzira za pulasitiki ya transcriptome yomwe imayambitsa OS pogwiritsa ntchito deta ya microarray ya nthawi, yomwe imatsanzira kugwiritsa ntchito ma OSM pochiza masamba a masamba a zomera za rosette m'zilombo zodya zomera. Kuchuluka kwa sampuli kumafanana ndi kuchuluka kwa pulasitiki komwe kumagwiritsidwa ntchito mu kafukufuku wa metabolomics (35). Poyerekeza ndi kusintha kwa metabolome komwe pulasitiki ya kagayidwe kachakudya imawonjezeka makamaka pakapita nthawi, timawona kuphulika kwa transient transcription m'masamba komwe kumachitika ndi Ms, komwe transcriptome inducibility (RDPI) ndi specialization (δj) zili pa 1 Panali kuwonjezeka kwakukulu kwa maola, ndipo kusiyana (Hj) panthawiyi, kufotokozera kwa BMP1 kunachepetsedwa kwambiri, kutsatiridwa ndi kupumula kwa specialization ya transcriptome (Chithunzi S4). Mabanja a majini a kagayidwe kachakudya (monga P450, glycosyltransferase, ndi BAHD acyltransferase) amatenga nawo mbali pakusonkhanitsa ma metabolites apadera kuchokera ku mayunitsi opangidwa kuchokera ku kagayidwe kachakudya koyambirira, kutsatira chitsanzo choyambirira cha high-specialization. Monga phunziro la chitsanzo, njira ya phenylalanine idasanthulidwa. Kusanthulaku kunatsimikizira kuti majini ofunikira mu kagayidwe ka phenolamide amayambitsidwa kwambiri ndi OS mu zomera zodya zomera poyerekeza ndi zomera zosakopeka, ndipo amagwirizana kwambiri ndi mawonekedwe awo. Cholembera cha MYB8 ndi majini opangidwa ndi PAL1, PAL2, C4H ndi 4CL omwe ali pamwamba pa njira iyi adawonetsa kuyambika koyambirira kwa kulembedwa. Acyltransferases zomwe zimagwira ntchito pakusonkhanitsa komaliza kwa phenolamide, monga AT1, DH29, ndi CV86, zikuwonetsa kukwera kwa nthawi yayitali (Chithunzi S4). Zomwe zawonedwa pamwambapa zikuwonetsa kuti kuyambika koyambirira kwa kudziwika kwa transcriptome ndi kukulitsa kwa kudziwika kwa metabolomics ndi njira yolumikizana, yomwe ikhoza kukhala chifukwa cha dongosolo lolamulira logwirizana lomwe limayambitsa yankho lamphamvu la chitetezo.
Kusinthidwa kwa zizindikiro za mahomoni a zomera kumagwira ntchito ngati gawo lolamulira lomwe limaphatikiza chidziwitso cha zomera kuti lisinthe kapangidwe ka thupi la zomera. Pambuyo pa kuyerekezera kwa herbivore, tinayesa kuchuluka kwa magulu ofunikira a mahomoni a zomera ndikuwona momwe nthawi imagwirizanirana pakati pawo [Pearson correlation coefficient (PCC)> 0.4] (Chithunzi 3A). Monga momwe timayembekezera, mahomoni a zomera okhudzana ndi biosynthesis amalumikizidwa mkati mwa netiweki ya co-expression ya mahomoni a zomera. Kuphatikiza apo, metabolic specificity (Si index) imayikidwa pa netiweki iyi kuti iwonetse mahomoni a zomera omwe amayambitsidwa ndi mankhwala osiyanasiyana. Madera awiri akuluakulu a herbivorous specific reaction amatengedwa: limodzi lili mu gulu la JA, komwe JA (mawonekedwe ake a biologically active JA-Ile) ndi zina zotumphukira za JA zimawonetsa Si score yapamwamba kwambiri; linalo ndi ethylene (ET). Gibberellin idawonetsa kuwonjezeka pang'ono kwa herbivore specificity, pomwe mahomoni ena a zomera, monga cytokinin, auxin, ndi abscisic acid, anali ndi induction specificity yochepa ya herbivores. Poyerekeza ndi kugwiritsa ntchito W + W yokha, kukulitsa kwa mtengo wapamwamba wa JA derivatives kudzera mu OS application (W + OS) kungasinthidwe kukhala chizindikiro champhamvu cha JAs. Mosayembekezereka, OSM ndi OSS1 zokhala ndi zinthu zosiyanasiyana zolimbikitsa zimadziwika kuti zimayambitsa kusonkhanitsa kofanana kwa JA ndi JA-Ile. Mosiyana ndi OSS1, OSM imayambitsidwa makamaka ndi ma OSM, pomwe OSS1 siyikulitsa kuyankha kwa mabala oyambira (Chithunzi 3B).
(A) Kusanthula kwa maukonde a co-expression kutengera kuwerengera kwa PCC kwa kuyerekezera kwa ma hormone a zomera omwe amayambitsidwa ndi herbivore. Node ikuyimira hormone imodzi ya chomera, ndipo kukula kwa node kumayimira chizindikiro cha Si chomwe chimadziwika ndi hormone ya chomera pakati pa chithandizo. (B) Kusonkhanitsa kwa JA, JA-Ile ndi ET m'masamba omwe amayambitsidwa ndi chithandizo chosiyana chomwe chikuwonetsedwa ndi mitundu yosiyanasiyana: apurikoti, W + OSM; buluu, W + OSSl; wakuda, W + W; imvi, C (kulamulira). Ma asterisks akuwonetsa kusiyana kwakukulu pakati pa chithandizo ndi kulamulira (ANOVA ya njira ziwiri kutsatiridwa ndi Tukey HSD post hoc multiple comparison, *** P <0.001). Kusanthula kwa chidziwitso cha (C)697 MS/MS (fayilo ya data S1) mu JA biosynthesis ndi impairment perception spectrum (irAOC ndi irCOI1) ndi (D)585 MS/MS (fayilo ya data S1) mu ETR1 yokhala ndi chizindikiro cha ET cholephera Mankhwala awiri oyeserera a herbivore adayambitsa mizere ya zomera ndi zomera zopanda kanthu zowongolera magalimoto (EV). Ma asterisks amasonyeza kusiyana kwakukulu pakati pa chithandizo cha W+OS ndi kulamulira kosawonongeka (ANOVA ya njira ziwiri kutsatiridwa ndi Tukey HSD post hoc multiple comparison, *P<0.05, **P<0.01 ndi ***P<0.001). (E) Ma graph ofalikira a kusagwirizana kosiyanasiyana ndi specialization. Mitundu ikuyimira mitundu yosiyanasiyana yosinthidwa majini; zizindikiro zikuyimira njira zosiyanasiyana zochizira: triangle, W + OSS1; rectangle, W + OSM; circle C
Kenako, timagwiritsa ntchito mtundu wa majini wa Nepenthes wochepetsedwa (irCOI1 ndi sETR1) m'magawo ofunikira a JA ndi ET biosynthesis (irAOC ndi irACO) ndi kuzindikira (irCOI1 ndi sETR1) kuti tiwunike kagayidwe ka mahomoni awiriwa a zomera pa zomera zomwe zimadya zomera. Mogwirizana ndi zoyeserera zam'mbuyomu, tatsimikizira kuyambitsa kwa herbivore-OS mu zomera zopanda kanthu (EV) (Chithunzi 3, C mpaka D) ndi kuchepa konse kwa Hj index komwe kumachitika chifukwa cha OSM, pomwe δj index idakwera. Yankho limaonekera kwambiri kuposa yankho lomwe lidayambitsidwa ndi OSS1. Chithunzi cha mizere iwiri pogwiritsa ntchito Hj ndi δj ngati ma coordinates chikuwonetsa kuchotsedwa kwapadera (Chithunzi 3E). Chizolowezi chodziwikiratu kwambiri ndichakuti m'mitundu yopanda chizindikiro cha JA, kusiyanasiyana kwa metabolome ndi kusintha kwapadera komwe kumachitika chifukwa cha zomera zomwe zimadya zomera kumachotsedwa pafupifupi kwathunthu (Chithunzi 3C). Mosiyana ndi zimenezi, kuona kwa ET chete m'zomera za sETR1, ngakhale kuti zotsatira zake zonse pa kusintha kwa kagayidwe kachakudya ka zomera zimakhala zochepa kwambiri kuposa za JA signaling, zimachepetsa kusiyana kwa zizindikiro za Hj ndi δj pakati pa kusangalatsa kwa OSM ndi OSS1 (Chithunzi 3D ndi Chithunzi S5). Izi zikusonyeza kuti kuwonjezera pa ntchito yaikulu ya JA signal transduction, ET signal transduction imagwiranso ntchito ngati kukonza bwino kagayidwe kachakudya ka mitundu ya zomera zodya zomera. Mogwirizana ndi ntchito yokonza bwinoyi, panalibe kusintha kulikonse mu metabolome inducibility m'zomera za sETR1. Kumbali ina, poyerekeza ndi zomera za sETR1, zomera za irACO zinayambitsa ma amplitude ofanana a kusintha kwa kagayidwe kachakudya komwe kumachitika chifukwa cha zomera zodya zomera, koma zinawonetsa kusiyana kwakukulu kwa Hj ndi δj pakati pa zovuta za OSM ndi OSS1 (Chithunzi S5).
Pofuna kupeza ma metabolites apadera omwe ali ndi gawo lofunikira pakuyankha kwa zamoyo za herbivores ndikukonza kupanga kwawo kudzera mu zizindikiro za ET, tidagwiritsa ntchito njira ya MS/MS yomwe idapangidwa kale. Njirayi imadalira njira ya bi-clustering kuti iwonetsenso banja la metabolic kuchokera ku MS/MS fragments [normalized dot product (NDP)] ndi similar score kutengera neutral loss (NL). Deta ya MS/MS yomwe idapangidwa kudzera mu kusanthula kwa mizere ya ET transgenic idapanga 585 MS/MS (data file S1), yomwe idathetsedwa powayika m'magulu asanu ndi awiri akuluakulu a MS/MS (M) (Chithunzi 4A). Ena mwa ma module awa ali ndi ma metabolites apadera omwe adadziwika kale: mwachitsanzo, M1, M2, M3, M4 ndi M7 ali ndi ma phenol derivatives osiyanasiyana (M1), flavonoid glycosides (M2), acyl shuga (M3 Ndi M4), ndi 17-HGL-DTG (M7). Kuphatikiza apo, chidziwitso cha kagayidwe kachakudya (Si index) cha metabolite imodzi mu module iliyonse chimawerengedwa, ndipo kugawa kwake kwa Si kumatha kuwoneka mwachilengedwe. Mwachidule, ma spectra a MS/MS omwe akuwonetsa kuchuluka kwa zomera ndi genotype specificity amadziwika ndi kuchuluka kwa Si, ndipo ziwerengero za kurtosis zikuwonetsa kugawa kwa ubweya pakona yakumanja. Kugawa kotereku kopanda mafuta kunapezeka mu M1, momwe phenol amide idawonetsa gawo lapamwamba kwambiri la Si (Chithunzi 4B). 17-HGL-DTG yomwe yatchulidwa kale ya herbivorous inducible mu M7 idawonetsa chigoli chapakati cha Si, kusonyeza mulingo wocheperako wa kusinthasintha pakati pa mitundu iwiri ya OS. Mosiyana ndi izi, ma metabolites ambiri apadera opangidwa, monga rutin, CGA, ndi acyl shuga, ndi ena mwa ma score otsika kwambiri a Si. Kuti tifufuze bwino zovuta za kapangidwe kake ndi kugawa kwa Si pakati pa ma metabolites apadera, netiweki yamamolekyulu idapangidwa pa module iliyonse (Chithunzi 4B). Kuneneratu kofunikira kwa chiphunzitso cha OD (komwe kwafotokozedwa mwachidule mu Chithunzi 1B) ndikuti kukonzanso kwa metabolites yapadera pambuyo pa herbivory kuyenera kuyambitsa kusintha kwa njira imodzi mu metabolites yokhala ndi chitetezo champhamvu, makamaka powonjezera kudziwika kwawo (mosiyana ndi kufalikira mwachisawawa) Mode) Metabolite yodzitchinjiriza yomwe yanenedweratu ndi chiphunzitso cha MT. Zambiri mwa zotumphukira za phenol zomwe zasonkhanitsidwa mu M1 zimagwirizana ndi kuchepa kwa magwiridwe antchito a tizilombo (32). Poyerekeza ma values ​​​​a Si mu metabolites a M1 pakati pa masamba oyambitsidwa ndi masamba omwe ali m'gulu la zomera zowongolera za EV pa maola 24, tidawona kuti kudziwika kwa metabolites ambiri pambuyo pa tizilombo tomera zomera kuli ndi chizolowezi chowonjezeka kwambiri (Chithunzi 4C). Kuwonjezeka kwapadera kwa mtengo wa Si kunapezeka kokha mu ma phenolamides odzitchinjiriza, koma palibe kuwonjezeka kwa mtengo wa Si komwe kunapezeka mu ma phenol ena ndi ma metabolites osadziwika omwe ali nawo mu gawoli. Iyi ndi chitsanzo chapadera, chomwe chikugwirizana ndi chiphunzitso cha OD. Maulosi akuluakulu a kusintha kwa kagayidwe kachakudya komwe kumachitika chifukwa cha herbivores ndi ofanana. Pofuna kuyesa ngati izi za phenolamide spectrum zinayambitsidwa ndi OS-specific ET, tinajambula metabolite Si index ndipo tinayambitsa kusiyana kwa mafotokozedwe pakati pa OSM ndi OSS1 mu genotypes za EV ndi sETR1 (Chithunzi 4D). Mu sETR1, kusiyana komwe kunayambitsidwa ndi phenamide pakati pa OSM ndi OSS1 kunachepetsedwa kwambiri. Njira yolumikizirana iwiri idagwiritsidwanso ntchito pa deta ya MS/MS yomwe idasonkhanitsidwa m'magulu omwe alibe JA yokwanira kuti azindikire ma module akuluakulu a MS/MS okhudzana ndi JA-regulated metabolic specialization (Chithunzi S6).
(A) Zotsatira za magulu a 585 MS/MS kutengera gawo logawana (NDP kufanana) ndi kutayika kwa neutral komwe kwagawidwa (NL kufanana) zimapangitsa kuti gawo (M) likhale logwirizana ndi banja lodziwika bwino la ma compound, kapena ndi kapangidwe ka Metabolite kosadziwika kapena kosakonzedwa bwino. Pafupi ndi gawo lililonse, kugawa kwa metabolite (MS/MS) specific (Si) kukuwonetsedwa. (B) Modular molecular network: Ma Nodes akuyimira MS/MS ndi m'mphepete, NDP (wofiira) ndi NL (buluu) MS/MS scores (cut-off,> 0.6). Chizindikiro cha metabolite specificity index (Si) chojambulidwa kutengera gawo (kumanzere) ndikujambulidwa ku netiweki ya molekyulu (kumanja). (C) Module M1 ya chomera cha EV mu constitutive (control) ndi induced state (simulated herbivore) pa maola 24: molecular network diagram (Si value ndi node size, defensive phenolamide imawonetsedwa mu buluu). (D) Chithunzi cha netiweki ya mamolekyu a M1 a mzere wa spectrum sETR1 wokhala ndi vuto la EV ndi ET: phenolic compound yomwe ikuimiridwa ndi node yozungulira yobiriwira, ndi kusiyana kwakukulu (P value) pakati pa chithandizo cha W + OSM ndi W + OSS1 monga kukula kwa Node. CP, N-caffeoyl-tyrosine; CS, N-caffeoyl-spermidine; FP, N-ferulic acid ester-uric acid; FS, N-ferulyl-spermidine; CoP, N', N “-Coumarolyl-tyrosine; DCS, N', N”-dicaffeoyl-spermidine; CFS, N', N”-caffeoyl, feruloyl-spermidine; Lycium barbarum in wolfberry Son; Nick. O-AS, shuga wa O-acyl.
Tinapitiliza kusanthula kuchokera ku genotype imodzi ya Nepenthes yomwe yachepa kufika ku mitundu yachilengedwe, komwe kusintha kwakukulu kwa intraspecific mu milingo ya JA yomera zomera ndi milingo yeniyeni ya metabolite kwafotokozedwa kale m'mitundu yachilengedwe (26). Gwiritsani ntchito deta iyi kuti muphatikize ma germplasm 43. Ma germplasm awa ali ndi mitundu 123 ya zomera kuchokera ku N. pallens. Zomera izi zinatengedwa kuchokera ku mbewu zomwe zinasonkhanitsidwa m'malo osiyanasiyana okhala ku Utah, Nevada, Arizona, ndi California (Chithunzi S7), tinawerengera kusiyanasiyana kwa metabolome (pano kumatchedwa kuchuluka kwa anthu) β kusiyanasiyana) ndi specialization yomwe imayambitsidwa ndi OSM. Mogwirizana ndi maphunziro am'mbuyomu, tinawona kusintha kwakukulu kwa kagayidwe kachakudya m'mbali mwa ma axes a Hj ndi δj, zomwe zikusonyeza kuti ma germplasm ali ndi kusiyana kwakukulu pakusinthasintha kwa mayankho awo a kagayidwe kachakudya ku zomera zodya zomera (Chithunzi S7). Bungweli likukumbutsa zomwe zidawonedwa kale za kusintha kwa JA komwe kumachitika chifukwa cha zomera zodya zomera, ndipo lakhala ndi mtengo wapamwamba kwambiri pa anthu amodzi (26, 36). Pogwiritsa ntchito JA ndi JA-Ile poyesa mgwirizano wonse pakati pa Hj ndi δj, tapeza kuti pali mgwirizano waukulu pakati pa JA ndi metabolome β diversity and specialization index (Chithunzi S7). Izi zikusonyeza kuti kusiyana kwa herbivore-induced induction mu JAs komwe kumapezeka pamlingo wa anthu kungakhale chifukwa cha ma polymorphisms ofunikira a kagayidwe kachakudya omwe amayambitsidwa ndi kusankha kuchokera ku tizilombo todya zomera.
Kafukufuku wakale wasonyeza kuti mitundu ya fodya imasiyana kwambiri pa kudalira kwa mitundu ndi kudalira kwa chitetezo cha kagayidwe kachakudya komwe kumayambitsa komanso komwe kumayambitsa matenda. Akukhulupirira kuti kusintha kumeneku pakufalitsa chizindikiro cha mankhwala oletsa udzu ndi mphamvu zodzitetezera kumayendetsedwa ndi kuchuluka kwa tizilombo, moyo wa zomera, komanso ndalama zopangira chitetezo m'malo omwe mtundu wina umakula. Tinaphunzira kusinthasintha kwa metabolome ya masamba yomwe imayambitsidwa ndi zomera za mitundu isanu ndi umodzi ya Nicotiana yochokera ku North America ndi South America. Mitundu iyi imagwirizana kwambiri ndi Nepenthes North America, yomwe ndi Nicolas Bociflo. La, N. nicotinis, Nicotiana n. udzu wochepetsedwa, Nicotiana tabacum, linear tobacco, fodya (Nicotiana spegazzinii) ndi fodya wa masamba a fodya (Nicotiana obtusifolia) (Chithunzi 5A) (37). Mitundu isanu ndi umodzi mwa iyi, kuphatikiza mitundu yodziwika bwino ya N. chonde, ndi zomera za pachaka za petunia clade, ndipo obtusifolia N. ndi zomera zosatha za sister clade Trigonophyllae (38). Pambuyo pake, W + W, W + OSM ndi W + OSS1 zinayambitsidwa pa mitundu isanu ndi iwiri iyi kuti aphunzire za kusintha kwa kagayidwe kachakudya ka mitundu ya tizilombo.
(A) Mtengo wa phylogenetic wa bootstrap wozikidwa pa kuthekera kwakukulu [kwa kapangidwe ka nuclear glutamine (38)] ndi kufalikira kwa mitundu isanu ndi iwiri yogwirizana ya Nicotiana (mitundu yosiyana) (37). (B) Chithunzi chofalikira cha kusiyanasiyana kwapadera kwa ma metabolic profiles a mitundu isanu ndi iwiri ya Nicotiana (939 MS/MS; fayilo ya data S1). Pa mulingo wa mitundu, kusiyanasiyana kwa metabolome kumagwirizana molakwika ndi mulingo wa specialization. Kusanthula kwa mgwirizano wa mitundu pakati pa kusiyanasiyana kwa kagayidwe kachakudya ndi specialization ndi JA accumulation kwawonetsedwa mu Chithunzi 2. S9. Mtundu, mitundu yosiyanasiyana; triangle, W + OSS1; rectangle, W + OSM; (C) Nicotiana JA ndi JA-Ile dynamics zayikidwa m'magulu malinga ndi amplitude ya OS excitation (two-way ANOVA ndi Tukey HSD post-multiple comparison, * P <0.05, ** P <0.01 ndi * ** Poyerekeza W + OS ndi W + W, P <0.001). Bokosi la (D) kusiyanasiyana ndi (E) kudziwika bwino kwa mtundu uliwonse pambuyo poyesa zomera ndi methyl JA (MeJA). Asterisk ikuwonetsa kusiyana kwakukulu pakati pa W + OS ndi W + W kapena lanolin kuphatikiza W (Lan + W) kapena Lan kuphatikiza MeJA (Lan + MeJa) ndi kulamulira kwa Lan (kusanthula kwa kusiyana kwa njira ziwiri, kutsatiridwa ndi kufananiza kwa Tukey kwa HSD post hoc multiple, *P<0.05, **P<0.01 ndi ***P<0.001).
Pogwiritsa ntchito njira ya dual cluster, tinapeza ma module 9 a 939 MS/MS (fayilo ya deta S1). Kapangidwe ka MS/MS komwe kamakonzedwanso ndi mankhwala osiyanasiyana kamasiyana kwambiri pakati pa ma module osiyanasiyana pakati pa mitundu (Chithunzi S8). Kuwona Hj (yomwe imatchedwa pano kuti γ-diversity ya mitundu) ndi δj kukuwonetsa kuti mitundu yosiyanasiyana imasonkhana m'magulu osiyanasiyana kwambiri m'malo osinthira kagayidwe kachakudya, komwe kugawikana kwa mitundu nthawi zambiri kumakhala kowonekera kwambiri kuposa kusangalatsa. Kupatula N. linear ndi N. obliquus, zimawonetsa mitundu yosiyanasiyana ya zotsatira zoyambitsa (Chithunzi 5B). Mosiyana ndi zimenezi, mitundu monga N. purpurea ndi N. obtusifolia ili ndi yankho losawoneka bwino la kagayidwe kachakudya ku chithandizo, koma metabolome ndi yosiyana kwambiri. Kugawa kwa mitundu yosiyanasiyana ya yankho la kagayidwe kachakudya komwe kumayambitsidwa kunabweretsa mgwirizano woipa pakati pa specialization ndi gamma diversity (PCC = -0.46, P = 4.9×10-8). Kusintha kwa milingo ya JA komwe kumabwera chifukwa cha OS kumagwirizana bwino ndi metabolome specialization, ndipo kumagwirizana molakwika ndi metabolic gamma diversity yomwe ikuwonetsedwa ndi mtundu uliwonse (Chithunzi 5B ndi Chithunzi S9). Ndikofunikira kudziwa kuti mitundu yomwe imatchulidwa kuti "signal response" mu Chithunzi 5C, monga Nepenthes nematodes, Nepenthes nepenthes, Nepenthes acute, ndi Nepenthes attenuated, idayambitsa zizindikiro zazikulu patatha mphindi 30. Kufalikira kwaposachedwa kwa OS-specific JA ndi JA-Ile, pomwe mabakiteriya ena otchedwa "signal responsive", monga Nepenthes mills, Nepenthes powdery ndi N. obtusifolia amangowonetsa JA-Ile Edge induction popanda OS specificity iliyonse (Chithunzi 5C). Pamlingo wa metabolic, monga tafotokozera pamwambapa, kwa Nepenthes attenuated, zinthu zomwe zimayankha chizindikiro zidawonetsa OS specific ndipo zidawonjezera kwambiri δj, pomwe zimachepetsa Hj. Mphamvu iyi ya OS-specific priming sinapezeke m'mitundu yomwe imagawidwa ngati signal non-reactive specifics (Chithunzi 5, D ndi E). Ma metabolite enieni a OS amagawidwa pafupipafupi pakati pa mitundu yoyankha zizindikiro, ndipo magulu a zizindikiro awa amasonkhana pamodzi ndi mitundu yomwe imayankha zizindikiro zochepa, pomwe mitundu yomwe imayankha zizindikiro zochepa imawonetsa kudalirana kochepa (Chithunzi S8). Zotsatirazi zikusonyeza kuti kulowetsedwa kwa ma JA enieni a OS ndi kusinthidwa kwapadera kwa metabolome yotsika kumalumikizidwa pamlingo wa mitundu.
Kenako, tinagwiritsa ntchito phala la lanolin lomwe lili ndi methyl JA (MeJA) pochiza zomera kuti tifufuze ngati njira zolumikizirazi zikulepheretsedwa ndi kupezeka kwa JA yomwe imagwiritsidwa ntchito ndi JA yakunja, yomwe idzakhala mu cytoplasm ya zomera. Kuwonongeka mwachangu ndi JA. Tapeza momwemonso kusintha pang'onopang'ono kuchokera ku mitundu yoyankha chizindikiro kupita ku mitundu yosayankha chizindikiro chifukwa cha kupezeka kosalekeza kwa JA (Chithunzi 5, D ndi E). Mwachidule, chithandizo cha MeJA chinasintha kwambiri metabolomes ya nematodes yolunjika, N. obliquus, N. aquaticus, N. pallens, ndi N. mikimotoi, zomwe zinapangitsa kuti δj ichuluke kwambiri komanso kuchepa kwa Hj. N. purpurea kunangowonetsa kuwonjezeka kwa δj, koma osati Hj. N. obtusifolia, yomwe yawonetsedwa kale kuti imasonkhanitsa milingo yotsika kwambiri ya JA, imayankhanso molakwika chithandizo cha MeJA pankhani ya kukonzanso metabolome. Zotsatirazi zikusonyeza kuti kupanga JA kapena kusintha kwa chizindikiro kumakhala kocheperako mwachilengedwe m'mitundu yosayankha chizindikiro. Kuti tiyese lingaliro ili, tinaphunzira mitundu inayi (N. pallens, N. mills, N. pink ndi N. microphylla) yoyambitsidwa ndi W + W, W + OSM ndi W + OSS1 Transcriptome (39). Mogwirizana ndi kapangidwe ka metabolome remodeling, mitunduyi imalekanitsidwa bwino mu transcriptome space, pakati pawo N. attenuated inawonetsa RDPI yapamwamba kwambiri yoyambitsidwa ndi OS, pomwe N. gracilis inali ndi yotsika kwambiri (Chithunzi 6A). Komabe, zidapezeka kuti kusiyanasiyana kwa transcriptome komwe kuyambitsidwa ndi N. oblonga kunali kotsika kwambiri pakati pa mitundu inayi, mosiyana ndi kusiyanasiyana kwakukulu kwa metabonomic kwa N. oblonga komwe kukuwonetsedwa kale m'mitundu isanu ndi iwiri. Kafukufuku wakale wasonyeza kuti gulu la majini okhudzana ndi zizindikiro zoyambirira za chitetezo, kuphatikiza zizindikiro za JA, limafotokoza kudziwika kwa mayankho oyambirira a chitetezo omwe ayambitsidwa ndi zoyambitsa zokhudzana ndi herbivore mu mitundu ya Nicotiana (39). Kuyerekeza njira zolumikizirana za JA pakati pa mitundu inayi iyi kunavumbula njira yosangalatsa (Chithunzi 6B). Majini ambiri omwe ali munjira iyi, monga AOC, OPR3, ACX ndi COI1, adawonetsa kuchuluka kwa kulowetsedwa kwa majini m'mitundu inayi iyi. Komabe, jini yofunika kwambiri, JAR4, imasintha JA kukhala mtundu wake wa JA-Ile wosungidwa m'thupi, ndipo kuchuluka kwake kolembedwa ndi kochepa kwambiri, makamaka mu N. mills, Nepenthes pieris ndi N. microphylla. Kuphatikiza apo, kulembedwa kwa jini lina la AOS kokha sikunapezeke mu N. bifidum. Kusintha kumeneku mu mawonekedwe a majini kungakhale komwe kumayambitsa mitundu yoopsa kwambiri yomwe imayamba chifukwa cha kupanga kochepa kwa JA mu mitundu ya zizindikiro komanso kulowetsedwa kwa N. gracilis.
(A) Kusanthula kwa chiphunzitso cha chidziwitso cha kukonzanso mayankho oyambirira a transcriptional a mitundu inayi ya fodya yogwirizana kwambiri yomwe idatengedwa mphindi 30 pambuyo pa kuyambitsa herbivory. RDPI imawerengedwa poyerekeza masamba omwe ayambitsidwa ndi herbivore OS ndi bala control. Mitunduyo imasonyeza mitundu yosiyanasiyana, ndipo zizindikirozo zimasonyeza njira zosiyanasiyana zochiritsira. (B) Kusanthula kwa kufotokozera kwa majini mu njira zolumikizirana za JA pakati pa mitundu inayi. Njira yosavuta ya JA ikuwonetsedwa pafupi ndi bokosi. Mitundu yosiyanasiyana imasonyeza njira zosiyanasiyana zochizira. Asterisk imasonyeza kuti pali kusiyana kwakukulu pakati pa chithandizo cha W + OS ndi W + W control (ya mayeso a t a Wophunzira a kusiyana kwa awiriawiri, *P<0.05, **P<0.01 ndi ***P<0.001). OPDA, 12-oxophytodienoic acid; OPC-8: 0,3-oxo-2(2′(Z)-pentenyl)-cyclopentane-1-octanoic acid.
Mu gawo lomaliza, tinaphunzira momwe kusintha kwa mitundu yosiyanasiyana ya tizilombo kwa metabolome ya mitundu yosiyanasiyana ya zomera kungakhalire kolimba ku zomera zodya udzu. Kafukufuku wakale adagogomezera mtundu wa Nicotiana. Kukana kwawo kwa Ms ndi mphutsi kumasiyana kwambiri (40). Pano, tinaphunzira kulumikizana pakati pa chitsanzo ichi ndi kusinthasintha kwawo kwa kagayidwe kachakudya. Pogwiritsa ntchito mitundu inayi ya fodya yomwe ili pamwambapa, ndikuyesera kulumikizana pakati pa kusiyanasiyana ndi kudziwika bwino kwa metabolome komwe kumachitika chifukwa cha zomera zodya udzu komanso kukana kwa zomera kwa Ms ndi Sl, tinapeza kuti kukana, kusiyanasiyana ndi kudziwika bwino kwa Sl All ndizogwirizana bwino, pomwe kulumikizana pakati pa kukana kwa akatswiri ndi kudziwika bwino ndikofooka, ndipo kulumikizana ndi kusiyanasiyana sikofunikira (Chithunzi S10). Ponena za kukana kwa S1, N. chinensis ndi N. gracilis zonse zomwe zachepetsedwa, zomwe zidawonetsedwa kale kuti zikuwonetsa milingo yonse ya JA signal transduction ndi metabolome plasticity, zinali ndi mayankho osiyana kwambiri ku kulowetsedwa kwa herbivore, ndipo zidawonetsanso kukana kofanana. Kugonana.
M'zaka makumi asanu ndi limodzi zapitazi, chiphunzitso cha chitetezo cha zomera chapereka dongosolo la chiphunzitso, kutengera momwe ofufuza adaneneratu kuchuluka kwa kusintha ndi ntchito za metabolites zapadera za zomera. Zambiri mwa ziphunzitsozi sizitsatira njira yachizolowezi ya malingaliro amphamvu (41). Amapereka maulosi ofunikira (3) pamlingo womwewo wa kusanthula. Pamene kuyesa maulosi ofunikira kulola kuti ziphunzitso zinazake zisanthuledwe, izi zipangitsa kuti gawolo lipite patsogolo. Thandizo, koma kanani ena (42). M'malo mwake, chiphunzitso chatsopanochi chimapanga maulosi pamlingo wosiyanasiyana wa kusanthula ndikuwonjezera gawo latsopano la malingaliro ofotokozera (42). Komabe, malingaliro awiri omwe aperekedwa pamlingo wa ntchito, MT ndi OD, amatha kufotokozedwa mosavuta ngati maulosi ofunikira a kusintha kwapadera kwa kagayidwe kachakudya komwe kumachitika chifukwa cha zinyama zodya zomera: chiphunzitso cha OD chimakhulupirira kuti kusintha kwa "malo" apadera a kagayidwe kachakudya kumakhala kolunjika kwambiri. Chiphunzitso cha MT chimakhulupirira kuti kusinthaku sikudzakhala kolunjika ndipo kudzakhala mwachisawawa m'malo a kagayidwe kachakudya, ndipo kumakhala ndi ma metabolites apamwamba kwambiri oteteza. Mayeso am'mbuyomu a maulosi a OD ndi MT ayesedwa pogwiritsa ntchito gulu laling'ono la mankhwala a "chitetezo" oyambirira. Mayeso awa a metabolite-centric amaletsa kuthekera kosanthula kukula ndi njira ya metabolome reconfiguration panthawi ya herbivory, ndipo salola mayeso mkati mwa dongosolo lokhazikika la ziwerengero kuti afune maulosi ofunikira omwe angaganizidwe onse. Kuyeza kusintha kwa metabolome ya zomera. Pano, tinagwiritsa ntchito ukadaulo watsopano mu metabolomics kutengera MS yowerengera ndipo tinachita deconvolution MS analysis mu ndalama zambiri zazidziwitso za chidziwitso kuti tiyese kusiyana pakati pa ziwiri zomwe zaperekedwa pamlingo wapadziko lonse wa metabolomics. Kuneneratu kwakukulu kwa chiphunzitsochi. Chiphunzitso cha chidziwitso chagwiritsidwa ntchito m'magawo ambiri, makamaka pankhani ya zamoyo zosiyanasiyana ndi kafukufuku woyendera zakudya (43). Komabe, monga momwe tikudziwira, iyi ndi ntchito yoyamba yogwiritsidwa ntchito pofotokoza malo azidziwitso za kagayidwe kachakudya a zomera ndikuthetsa mavuto azachilengedwe okhudzana ndi kusintha kwakanthawi kwa kagayidwe kachakudya poyankha zizindikiro zachilengedwe. Makamaka, kuthekera kwa njira iyi kuli mu kuthekera kwake kuyerekeza mapangidwe mkati ndi pakati pa mitundu ya zomera kuti tiwone momwe zamoyo zamasamba zasinthira kuchokera ku mitundu yosiyanasiyana kupita ku mitundu yosiyanasiyana ya macroevolutionary pamlingo wosiyana wa kusintha. Kagayidwe kachakudya.
Kusanthula kwa zigawo zazikulu (PCA) kumasintha deta ya multivariate kukhala malo ochepetsera kukula kwa deta kuti njira yayikulu ya detayo ifotokozedwe, kotero nthawi zambiri imagwiritsidwa ntchito ngati njira yofufuzira kuti iwonetse deta, monga deconvolution metabolome. Komabe, kuchepetsa kukula kwa deta kudzataya gawo la zomwe zili mu deta, ndipo PCA singathe kupereka zambiri zokhudzana ndi makhalidwe omwe ali okhudzana kwambiri ndi chiphunzitso cha zachilengedwe, monga: momwe zomera zimasinthira kusiyanasiyana m'magawo apadera (monga kulemera, kugawa) Ndi kuchuluka) metabolites? Ndi metabolites ziti zomwe zimaneneratu momwe chomera china chimakhalira? Kuchokera pamalingaliro a kudziwika, kusiyanasiyana, ndi kusasinthika, zomwe zili mu metabolite yeniyeni ya masamba zimawonongeka, ndipo zimapezeka kuti kudya zomera kumatha kuyambitsa kagayidwe kachakudya kena. Mosayembekezereka, tinaona kuti, monga momwe tafotokozera mu zizindikiro za chidziwitso zomwe zagwiritsidwa ntchito, mkhalidwe wa kagayidwe kachakudya womwe umachitika umakhala wofanana kwambiri pambuyo pa kuukira kwa nyama ziwiri zodya udzu (wodyetsa usiku Sl) ndi katswiri wa Solanaceae Ms. Ngakhale kuti khalidwe lawo lodya ndi kuchuluka kwawo ndizosiyana kwambiri. Woyambitsa mafuta acid-amino acid conjugate (FAC) mu OS (31). Pogwiritsa ntchito herbivore OS pochiza mabala obowoka, chithandizo chofanana cha herbivore chinawonetsanso zomwezo. Njira yokhazikika iyi yotsanzira momwe zomera zimayankhira ku ziwopsezo za herbivore imachotsa zinthu zosokoneza zomwe zimachitika chifukwa cha kusintha kwa machitidwe odya a nyama zodya udzu, zomwe zimapangitsa kuti pakhale kuwonongeka kosiyanasiyana nthawi zosiyanasiyana (34). FAC, yomwe imadziwika kuti ndiyo chifukwa chachikulu cha OSM, imachepetsa mayankho a JAS ndi mahomoni ena a zomera mu OSS1, pomwe OSS1 imachepetsa nthawi mazana ambiri (31). Komabe, OSS1 idayambitsa kuchuluka kofanana kwa JA poyerekeza ndi OSM. Zawonetsedwa kale kuti yankho la JA mu Nepenthes yofooka limakhala lovuta kwambiri ku OSM, komwe FAC imatha kusunga ntchito yake ngakhale itachepetsedwa ndi madzi 1:1000 (44). Chifukwa chake, poyerekeza ndi OSM, ngakhale FAC mu OSS1 ndi yotsika kwambiri, ndiyokwanira kuyambitsa kufalikira kwa JA kokwanira. Kafukufuku wakale wasonyeza kuti mapuloteni ofanana ndi porin (45) ndi oligosaccharides (46) angagwiritsidwe ntchito ngati zizindikiro za mamolekyulu kuti ayambe kuyankhidwa kwa chitetezo cha zomera mu OSS1. Komabe, sizikudziwikabe ngati zolimbikitsa izi mu OSS1 ndizo zimayambitsa kusonkhanitsa kwa JA komwe kwawonedwa mu kafukufuku wapano.
Ngakhale kuti pali maphunziro ochepa omwe akufotokoza zala zosiyanasiyana zomwe zimachitika chifukwa cha kugwiritsa ntchito zomera zosiyanasiyana kapena JA kapena SA (salicylic acid) (47), palibe amene wasokoneza kusokonezeka kwa mitundu yosiyanasiyana ya zomera zomwe zimamera mu netiweki ya udzu wa zomera ndi zotsatira zake pa zomwe zimachitika. Kusanthula kumeneku kunatsimikiziranso kuti kulumikizana kwa ma hormone amkati ndi mahomoni ena a zomera kupatula ma JA kumapanga kulunjika kwa kagayidwe kachakudya komwe kumachitika chifukwa cha zomera zomwe zimamera. Makamaka, tinapeza kuti ET yomwe imayambitsidwa ndi OSM inali yayikulu kwambiri kuposa yomwe imayambitsidwa ndi OSS1. Njirayi ikugwirizana ndi kuchuluka kwa FAC mu OSM, komwe ndi kofunikira komanso kokwanira kuyambitsa kuphulika kwa ET (48). Pankhani yolumikizana pakati pa zomera ndi zomera zomwe zimamera, ntchito yodziwitsa ya ET pa metabolite ya zomera idakali yokhazikika ndipo imangoyang'ana gulu limodzi lokha. Kuphatikiza apo, maphunziro ambiri agwiritsa ntchito kugwiritsa ntchito ET kapena zinthu zake zoyambira kapena zoletsa zosiyanasiyana kuti aphunzire za kayendetsedwe ka ET, zomwe pakati pa izi mankhwala ochokera kunja amabweretsa zotsatirapo zambiri zosafunikira. Monga momwe tikudziwira, kafukufukuyu akuyimira kafukufuku woyamba waukulu wa udindo wa ET pakugwiritsa ntchito ET popanga ndi kuzindikira zomera zosinthika kuti zigwirizane ndi metabolome dynamics ya zomera. Kulowetsedwa kwa ET kwa herbivore-specific ET kumatha kusintha momwe metabolome imayankhira. Chofunika kwambiri ndi kusintha kwa majini a ET biosynthesis (ACO) ndi perception (ETR1) omwe adavumbulutsa kuchuluka kwa phenolamides kwa herbivore-specific de novo. Zawonetsedwa kale kuti ET imatha kusintha kuchuluka kwa nicotine komwe kumabwera chifukwa cha JA poyang'anira putrescine N-methyltransferase (49). Komabe, kuchokera kumalingaliro a makina, sizikudziwika bwino momwe ET imasinthira kulowetsedwa kwa phenamide. Kuwonjezera pa ntchito yotumiza chizindikiro cha ET, kagayidwe kachakudya kangasinthidwenso ku S-adenosyl-1-methionine kuti kayendetse ndalama zomwe zimagwiritsidwa ntchito mu polyaminophenol amides. S-adenosyl-1-methionine ndi ET ndipo ndi njira yodziwika bwino ya polyamine biosynthetic pathway. Njira yomwe chizindikiro cha ET chimayendetsera kuchuluka kwa phenolamide ikufunika kuphunziridwa kwambiri.
Kwa nthawi yayitali, chifukwa cha kuchuluka kwa ma metabolites apadera a kapangidwe kosadziwika, chidwi chachikulu pamagulu enaake a kagayidwe kachakudya sichinathe kuwunika mosamala kusintha kwa nthawi kwa kusiyanasiyana kwa kagayidwe kachakudya pambuyo pa kuyanjana kwa zamoyo. Pakadali pano, kutengera kusanthula kwa chidziwitso, zotsatira zazikulu za MS/MS spectrum acquisition kutengera ma metabolites osakondera ndikuti ma herbivores omwe amadya kapena kutsanzira ma herbivores amapitiliza kuchepetsa kusiyanasiyana konse kwa kagayidwe kachakudya kwa masamba a metabolome pomwe akuwonjezera digiri yake yaukadaulo. Kuwonjezeka kwakanthawi kwa metabolome specificity komwe kumachitika chifukwa cha ma herbivores kumalumikizidwa ndi kuwonjezeka kwa mgwirizano mu transcriptome specificity. Mbali yomwe imathandizira kwambiri pa metabolome specialization yayikuluyi (yokhala ndi mtengo wapamwamba wa Si) ndi metabolite yapadera yokhala ndi ntchito yodziwika kale ya udzu. Chitsanzochi chikugwirizana ndi kulosera kwa chiphunzitso cha OD, koma kulosera kwa MT kokhudzana ndi kusakhazikika kwa metabolome reprogramming sikofanana. Komabe, deta iyi ikugwirizananso ndi kulosera kwa mtundu wosakanikirana (MT yabwino kwambiri; Chithunzi 1B), chifukwa ma metabolites ena osadziwika omwe ali ndi ntchito zodzitetezera zosadziwika akhoza kutsatirabe kugawa kwa Si mwachisawawa.
Chitsanzo chodziwika bwino chomwe chapezeka mu kafukufukuyu ndichakuti kuyambira pamlingo wa kusintha kwa zinthu zazing'ono (chiwerengero cha zomera chimodzi ndi fodya) mpaka pamlingo waukulu wa kusintha (mitundu ya fodya yogwirizana kwambiri), magawo osiyanasiyana a dongosolo la kusintha kwa zinthu ali mu "chitetezo chabwino kwambiri". Pali kusiyana kwakukulu pa luso la nyama zodya zomera. Moore et al. (20) ndi Kessler ndi Kalske (1) adapereka lingaliro lodziyimira pawokha kuti asinthe magawo atatu ogwira ntchito a zamoyo zosiyanasiyana omwe poyamba adasiyanitsidwa ndi Whittaker (50) kukhala kusintha kwa nthawi komwe kumayambitsidwa ndi kuyambitsidwa kwa mitundu yosiyanasiyana ya mankhwala; olemba awa sanafotokoze mwachidule. Njira zosonkhanitsira deta yayikulu ya metabolome sizikufotokozanso momwe mungawerengere kusiyana kwa kagayidwe kachakudya kuchokera ku deta iyi. Mu kafukufukuyu, kusintha pang'ono ku gulu la ntchito la Whittaker kudzaganizira kusiyana kwa kagayidwe kachakudya monga kusiyana kwa ma spectra a MS/MS mu chomera china, ndi kusiyana kwa β-metabolic monga kagayidwe kachakudya ka intraspecific ka gulu la anthu Space, ndi kusiyana kwa γ-metabolic kudzakhala kuwonjezera kusanthula kwa mitundu yofanana.
Chizindikiro cha JA n'chofunikira kwambiri pa mayankho osiyanasiyana a kagayidwe ka nyama zodya udzu. Komabe, palibe kuyesa kokwanira kwa kuchuluka kwa momwe JA imagwirira ntchito mkati mwa biosynthesis ku kusiyanasiyana kwa metabolome, ndipo ngati chizindikiro cha JA ndi malo ofunikira kwambiri pakusiyana kwa kagayidwe ka nyama koyambitsidwa ndi nkhawa pamlingo wapamwamba wa kusintha kwakukulu sikukudziwikabe. Tawona kuti chikhalidwe cha Nepenthes herbivorous chodya udzu chimayambitsa kudziwika bwino kwa metabolome ndipo kusiyanasiyana kwa kudziwika bwino kwa metabolome mkati mwa anthu a mitundu ya Nicotiana komanso pakati pa mitundu yogwirizana kwambiri ya Nicotiana kumagwirizana bwino ndi JA signaling. Kuphatikiza apo, chizindikiro cha JA chikasokonekera, kudziwika kwa kagayidwe ka nyama komwe kumachitika chifukwa cha genotype imodzi ya herbivore kudzathetsedwa (Chithunzi 3, C ndi E). Popeza kusintha kwa kagayidwe ka nyama kwa anthu a Nepenthes omwe achepetsedwa mwachilengedwe kumakhala kwakukulu, kusintha kwa kusiyanasiyana kwa β ndi kudziwika kwake mu kusanthula uku kungayambitsidwe kwambiri ndi kukondweretsedwa kwakukulu kwa magulu azinthu zolemera za metabolite. Magulu azinthu awa amalamulira gawo la mbiri ya metabolome ndipo amabweretsa mgwirizano wabwino ndi zizindikiro za JA.
Popeza njira zama biochemical za mitundu ya fodya yogwirizana nayo ndi yosiyana kwambiri, ma metabolites amadziwika bwino mu gawo la qualitative, kotero ndi yowunikira kwambiri. Kukonza kwa chidziwitso cha mbiri ya kagayidwe kachakudya komwe kwagwidwa kukuwonetsa kuti kulowetsedwa kwa zomera kumawonjezera malonda pakati pa kusiyanasiyana kwa gamma ya metabolic ndi specialization. Chizindikiro cha JA chimagwira ntchito yayikulu mu malonda awa. Kuwonjezeka kwa metabolome specialization kukugwirizana ndi kulosera kwakukulu kwa OD ndipo kumagwirizana bwino ndi chizindikiro cha JA, pomwe chizindikiro cha JA chikugwirizana molakwika ndi kusiyanasiyana kwa gamma ya metabolic. Ma model awa akuwonetsa kuti mphamvu ya OD ya zomera imatsimikiziridwa makamaka ndi plasticity ya JA, kaya pamlingo wa microevolutionary kapena pamlingo waukulu wa chisinthiko. Kuyesa kwa JA komwe kumaletsa zolakwika za JA biosynthesis kukuwonetsanso kuti mitundu ya fodya yogwirizana kwambiri imatha kusiyanitsa mitundu yogwirizana ndi zizindikiro ndi zosayankha zizindikiro, monga momwe JA yawo imagwirira ntchito komanso metabolome plasticity yoyambitsidwa ndi zomera. Mitundu yosayankha zizindikiro singathe kuyankha chifukwa cholephera kupanga JA yachilengedwe ndipo chifukwa chake imakhala ndi zoletsa zathupi. Izi zitha kuchitika chifukwa cha kusintha kwa majini ena ofunikira mu njira yolumikizirana ya JA (AOS ndi JAR4 mu N. crescens). Zotsatirazi zikuwonetsa kuti machitidwe a kusintha kwa macroevolutionary awa amatha kuyendetsedwa makamaka ndi kusintha kwa kuzindikira kwa mahomoni amkati ndi kuyankha.
Kuwonjezera pa kuyanjana pakati pa zomera ndi zomera zodya zomera, kufufuza kusiyanasiyana kwa kagayidwe kachakudya kukugwirizana ndi kupita patsogolo konse kofunikira kwa chiphunzitso pakufufuza za kusintha kwa zamoyo ku chilengedwe ndi kusintha kwa makhalidwe ovuta a phenotypic. Ndi kuwonjezeka kwa kuchuluka kwa deta yomwe yapezedwa ndi zida zamakono za MS, kuyesa kwa malingaliro pa kusiyanasiyana kwa kagayidwe kachakudya tsopano kumatha kupitirira kusiyana kwa metabolite ya munthu aliyense/gulu ndikuchita kusanthula kwapadziko lonse lapansi kuti awulule machitidwe osayembekezereka. Mu ndondomeko ya kusanthula kwakukulu, fanizo lofunika ndi lingaliro loganiza mamapu ofunikira omwe angagwiritsidwe ntchito kufufuza deta. Chifukwa chake, zotsatira zofunika za kuphatikiza kwa metabolomics ya MS/MS yopanda tsankho ndi chiphunzitso cha chidziwitso ndikuti imapereka njira yosavuta yomwe ingagwiritsidwe ntchito kupanga mamapu kuti ayang'ane kusiyanasiyana kwa kagayidwe kachakudya pamiyeso yosiyanasiyana ya taxonomic. Ndi chofunikira chachikulu cha njira iyi. Kuphunzira za kusintha kwa micro/macro ndi chilengedwe cha anthu ammudzi.
Pa mlingo wa kusintha kwakukulu, mfundo yaikulu ya chiphunzitso cha kusintha kwa zomera ndi tizilombo cha Ehrlich ndi Raven (51) ndikulosera kuti kusiyana kwa kusiyana kwa kagayidwe kachakudya pakati pa mitundu ya zomera ndiko chifukwa cha kusiyanasiyana kwa mizere ya zomera. Komabe, m'zaka makumi asanu kuchokera pamene ntchito yofunikayi inafalitsidwa, lingaliro ili silinayesedwe kawirikawiri (52). Izi makamaka chifukwa cha makhalidwe a phylogenetic a makhalidwe ofanana a kagayidwe kachakudya m'mizere yayitali ya zomera. Kusowa kumeneku kungagwiritsidwe ntchito kulimbitsa njira zowunikira cholinga. Kayendedwe ka MS/MS komwe kakuchitika pano komwe kamakonzedwa ndi chiphunzitso cha chidziwitso kumayesa kufanana kwa kapangidwe ka MS/MS kwa ma metabolites osadziwika (popanda kusankha metabolite kale) ndikusintha ma MS/MS awa kukhala gulu la MS/MS, motero mu metabolism yaukadaulo Ma model awa a kusintha kwakukulu amayerekezeredwa mu sikelo yogawa. Zizindikiro zosavuta za ziwerengero. Njirayi ndi yofanana ndi kusanthula kwa phylogenetic, komwe kungagwiritse ntchito kulinganiza kwa sequence kuti kuwerengetse kuchuluka kwa kusiyanasiyana kapena kusintha kwa khalidwe popanda kulosera kale.
Pa mlingo wa biochemical, lingaliro la Firn ndi Jones (53) lofufuza likuwonetsa kuti kusiyanasiyana kwa kagayidwe kachakudya kumasungidwa pamlingo wosiyana kuti kupereke zinthu zopangira kuti zigwire ntchito zamoyo za metabolites zomwe sizinali zogwirizana kapena zosinthidwa. Njira za chiphunzitso cha chidziwitso zimapereka chimango momwe kusintha kwa metabolite komwe kumachitika panthawi yodziwitsa metabolite kumatha kuwerengedwa ngati gawo la njira yowunikira yomwe ikuperekedwa: kusintha kwa biologically kuchokera ku specificity yochepa kupita ku specificity yayikulu Ma metabolites oletsedwa a malo enaake.
Mwachidule, m'masiku oyambirira a sayansi ya zamoyo zamamolekyu, ziphunzitso zofunika zoteteza zomera zinapangidwa, ndipo njira zoganizira mozama zomwe zimayendetsedwa ndi maganizo zimaonedwa kuti ndi njira yokhayo yopitira patsogolo sayansi. Izi zimachitika makamaka chifukwa cha zofooka zaukadaulo poyesa metabolome yonse. Ngakhale njira zoganizira mozama ndizothandiza kwambiri posankha njira zina zoyambitsa, kuthekera kwawo kupititsa patsogolo kumvetsetsa kwathu kwa maukonde a biochemical kuli kochepa kuposa njira zowerengera zomwe zilipo mu sayansi yamakono yogwiritsa ntchito deta yambiri. Chifukwa chake, ziphunzitso zomwe sizinganenedweratu zili kutali kwambiri ndi zomwe zilipo, kotero njira yoganizira/yoyesera yopita patsogolo m'munda wofufuza singathe kuthetsedwa (4). Tikuwona kuti njira yowerengera ya metabolomics yomwe yayambitsidwa pano ikhoza kuyambitsanso chidwi ndi nkhani zaposachedwa (momwe) komanso zomaliza (chifukwa) za kusiyanasiyana kwa kagayidwe kachakudya, ndikuthandizira ku nthawi yatsopano ya sayansi yowongolera deta. Nthawiyi idayang'ananso ziphunzitso zofunika zomwe zidalimbikitsa mibadwo yakale.
Kudyetsa zomera mwachindunji kumachitika mwa kukweza mphutsi yachiwiri kapena mphutsi ya Sl pa tsamba limodzi loyera la chomera chimodzi chotulutsa duwa, ndi zomera 10 zofanana pa chomera chilichonse. Mphutsi za tizilombo zinamangiriridwa ndi ma clamp, ndipo minofu yotsala ya tsamba inasonkhanitsidwa maola 24 ndi 72 pambuyo pa matenda ndipo inazizira mofulumira, ndipo zinthu zomwe zinapangidwazo zinachotsedwa.
Yerekezerani mankhwala ophera zomera mwanjira yogwirizana kwambiri. Njirayi ndikugwiritsa ntchito mawilo a nsalu kuti muboole mizere itatu ya minga mbali zonse ziwiri za nthiti za masamba atatu okulirakulira a chomera panthawi yomwe chomeracho chikukulirakulira, ndipo nthawi yomweyo ikani 1:5 Diluted Ms. Kapena gwiritsani ntchito zala zotetezedwa kuti muike S1 OS mu bala loboola. Dulani ndi kukonza tsamba monga tafotokozera pamwambapa. Gwiritsani ntchito njira yomwe yafotokozedwa kale kuti muchotse ma metabolites oyambira ndi mahomoni a zomera (54).
Pa ntchito za JA zakunja, masamba atatu a petiole a chomera chilichonse mwa zisanu ndi chimodzi cha maluwa a mtundu uliwonse amathiridwa ndi 20μl ya lanolin phala yokhala ndi 150μg MeJA (Lan + MeJA), ndi 20μl ya lanolin kuphatikiza mankhwala a mabala (Lan + W), kapena gwiritsani ntchito 20μl ya lanolin yoyera ngati chowongolera. Masambawo adakololedwa patatha maola 72 kuchokera pamene adachiritsidwa, adasungidwa mu nayitrogeni yamadzimadzi, ndikusungidwa pa -80°C mpaka atagwiritsidwa ntchito.
Mizere inayi ya JA ndi ET transgenic, yomwe ndi irAOC (36), irCOI1 (55), irACO ndi sETR1 (48), yapezeka mu gulu lathu lofufuza. IrAOC yawonetsa kwambiri kuchepa kwa milingo ya JA ndi JA-Ile, pomwe irCOI1 sinali yogwirizana ndi JAs. Poyerekeza ndi EV, kuchuluka kwa JA-Ile kunawonjezeka. Mofananamo, irACO idzachepetsa kupanga kwa ET, ndipo poyerekeza ndi EV, sETR1, yomwe siigwirizana ndi ET, idzawonjezera kupanga kwa ET.
Choyezera kuwala kwa laser (Sensor Sense ET-300 real-time ET sensor) chimagwiritsidwa ntchito poyezera ET mosalowerera. Nthawi yomweyo chithandizo chitatha, theka la masamba linadulidwa ndikusamutsidwira ku botolo lagalasi lotsekedwa la 4-ml, ndipo mutu wake unaloledwa kusonkhana mkati mwa maola 5. Pa nthawi yoyezera, botolo lililonse linatsukidwa ndi madzi a malita awiri pa ola limodzi a mpweya woyera kwa mphindi 8, omwe kale adadutsa mu chothandizira choperekedwa ndi Sensor Sense kuti achotse CO2 ndi madzi.
Deta ya microarray idasindikizidwa koyamba mu (35) ndipo idasungidwa mu National Center for Biotechnology Information (NCBI) Gene Expression Comprehensive Database (nambala yolowera GSE30287). Deta yofanana ndi masamba omwe adayambitsidwa ndi chithandizo cha W + OSMs ndi ulamuliro wosawonongeka idachotsedwa pa kafukufukuyu. Mphamvu yaiwisi ndi log2. Asanafufuze ziwerengero, maziko adasinthidwa ndikusinthidwa kukhala ofanana ndi 75th percentile pogwiritsa ntchito pulogalamu ya R.
Deta yoyambirira ya RNA sequencing (RNA-seq) ya mitundu ya Nicotiana idatengedwa kuchokera ku NCBI Short Reading Archives (SRA), nambala ya polojekitiyi ndi PRJNA301787, yomwe idanenedwa ndi Zhou et al. (39) ndipo ipitilira monga momwe tafotokozera mu (56). Deta yosaphika yomwe idakonzedwa ndi W + W, W + OSM ndi W + OSS1 yofanana ndi mitundu ya Nicotiana idasankhidwa kuti isanthulidwe mu kafukufukuyu, ndipo idakonzedwa motere: Choyamba, kuwerenga kosaphika kwa RNA-seq kudasinthidwa kukhala mtundu wa FASTQ. HISAT2 imasintha FASTQ kukhala SAM, ndipo SAMtools imasintha mafayilo a SAM kukhala mafayilo a BAM osankhidwa. StringTie imagwiritsidwa ntchito kuwerengera kufotokozera kwa majini, ndipo njira yake yofotokozera ndi yakuti pali zidutswa pa zidutswa zikwizikwi pa zidutswa miliyoni zotsatizana zolembera.
Chigawo cha Acclaim chromatographic (150 mm x 2.1 mm; kukula kwa tinthu 2.2μm) chomwe chimagwiritsidwa ntchito pofufuza ndi chigawo choteteza cha 4 mm x 4 mm chimakhala ndi zinthu zomwezo. Chigawo chotsatirachi cha binary gradient chikugwiritsidwa ntchito mu dongosolo la Dionex UltiMate 3000 Ultra High Performance Liquid Chromatography (UHPLC): mphindi 0 mpaka 0.5, isocratic 90% A [madzi osungunuka, 0.1% (v/v) acetonitrile ndi 0.05% formic acid], 10% B (Acetonitrile ndi 0.05% formic acid); mphindi 0.5 mpaka 23.5, gawo la gradient ndi 10% A ndi 90% B, motsatana; mphindi 23.5 mpaka 25, isocratic 10% A ndi 90% B. Kuthamanga kwa madzi ndi 400μl/min. Pa kusanthula konse kwa MS, ikani column eluent mu quadrupole ndi time-of-flight (qTOF) analyzer yokhala ndi electrospray source yomwe imagwira ntchito mu positive ionization mode (capillary voltage, 4500 V; capillary outlet 130 V; Kuuma kutentha 200°C; kuumitsa mpweya malita 10/min).
Chitani kusanthula kwa zidutswa za MS / MS (pano komwe kumatchedwa MS / MS) komwe sikungasiyanitsidwe ndi deta kuti mupeze zambiri zokhudza kapangidwe kake ka kagayidwe kachakudya komwe kangadziwike. Lingaliro la njira yosankhira ya MS/MS limadalira mfundo yakuti quadrupole ili ndi zenera lalikulu kwambiri lodzipatula la mass [chifukwa chake, ganizirani zizindikiro zonse za mass-to-charge ratio (m/z) ngati zidutswa]. Pachifukwa ichi, chifukwa chida cha Impact II sichinathe kupanga CE tilt, kusanthula kodziyimira pawokha kunachitika pogwiritsa ntchito ma values ​​​​owonjezeka a collision-induced dissociation collision energy (CE). Mwachidule, choyamba santhulani chitsanzocho ndi UHPLC-electrospray ionization/qTOF-MS pogwiritsa ntchito single mass spectrometry mode (mikhalidwe yocheperako yopangidwa ndi in-source fragmentation), kusanthula kuyambira m/z 50 mpaka 1500 pafupipafupi yobwerezabwereza ya 5 Hz. Gwiritsani ntchito nayitrogeni ngati mpweya wogundana posanthula MS/MS, ndipo chitani miyeso yodziyimira payokha pa ma voltage anayi osiyanasiyana otsatirawa: 20, 30, 40, ndi 50 eV. Munthawi yonse yoyezera, quadrupole ili ndi zenera lalikulu kwambiri lodzipatula, kuyambira m/z 50 mpaka 1500. Pamene kuyesa kwa m/z ndi m'lifupi mwa thupi lakutsogolo kwayikidwa pa 200, kuchuluka kwa kulemera kumayatsidwa zokha ndi pulogalamu yogwiritsira ntchito ya chidacho ndi 0 Da. Sakani zidutswa za kulemera monga momwe zilili mu single mass mode. Gwiritsani ntchito sodium formate (50 ml isopropanol, 200 μl formic acid ndi 1 ml 1M NaOH aqueous solution) kuti muwongolere kulemera. Pogwiritsa ntchito algorithm ya Bruker yolondola kwambiri, fayilo ya deta imawongoleredwa pambuyo poyendetsa avareji ya spectrum munthawi inayake. Gwiritsani ntchito ntchito yotumizira kunja ya pulogalamu ya Data Analysis v4.0 (Brook Dalton, Bremen, Germany) kuti musinthe mafayilo a data osaphika kukhala mtundu wa NetCDF. Deta ya MS/MS yasungidwa mu database yotseguka ya metabolomics MetaboLights (www.ebi.ac.uk) ndi nambala yolowera. MTBLS1471.
Kusonkhana kwa MS/MS kumatha kuchitika kudzera mu kusanthula kwa ubale pakati pa zizindikiro za MS1 ​​ndi MS/MS kuti pakhale mphamvu zochepa komanso zazikulu zogundana komanso malamulo atsopano omwe agwiritsidwa ntchito. Chikalata cha R chimagwiritsidwa ntchito kuzindikira kusanthula kwa ubale wa kugawa kwa precursor ku chinthucho, ndipo chikalata cha C# (https://github.com/MPI-DL/indiscriminant-MS-MS-assembly-pipeline) chimagwiritsidwa ntchito kukhazikitsa malamulowo.
Pofuna kuchepetsa zolakwika zabodza zomwe zimachitika chifukwa cha phokoso lakumbuyo ndi kulumikizana kwabodza komwe kumachitika chifukwa chozindikira zinthu zina za m/z m'zitsanzo zochepa chabe, timagwiritsa ntchito ntchito ya "filled peak" ya phukusi la R XCMS (pokonza phokoso lakumbuyo). Iyenera kugwiritsidwa ntchito m'malo mwa mphamvu ya "NA" (yosapezeka). Ntchito ya fill peak ikagwiritsidwa ntchito, pamakhalabe mphamvu zambiri za "0″ mu data set zomwe zingakhudze kuwerengera kwa mgwirizano. Kenako, timayerekeza zotsatira zokonza deta zomwe zapezeka pamene ntchito ya filled peak ikagwiritsidwa ntchito komanso pamene ntchito ya filled peak ikagwiritsidwa ntchito, ndikuwerengera phindu la phokoso lakumbuyo kutengera mtengo woyesedwa wokonzedwa, kenako ndikusinthira mitengo ya 0 intensity iyi ndi mtengo wakumbuyo wowerengedwa. Tinaganiziranso zinthu zomwe mphamvu yake idapitilira katatu kuposa mtengo wakumbuyo ndipo timaziona ngati "peaks zenizeni." Pakuwerengera kwa PCC, zizindikiro za m/z zokha za sample precursor (MS1) ndi fragment data sets zokhala ndi peaks zosachepera zisanu ndi zitatu zenizeni zimaganiziridwa.
Ngati mphamvu ya mawonekedwe a precursor quality mu chitsanzo chonse ikugwirizana kwambiri ndi mphamvu yocheperako ya mawonekedwe omwewo omwe amakhudzidwa ndi mphamvu yochepa kapena yayikulu yogundana, ndipo mawonekedwewo sanatchulidwe kuti ndi peak ya isotope ndi CAMERA, akhoza kufotokozedwanso. Kenako, powerengera mawiri onse omwe angakhalepo a precursor-product mkati mwa masekondi atatu (nthawi yoyerekeza yosungira kuti asunge kwambiri), kusanthula kwa mgwirizano kumachitika. Pokhapokha ngati mtengo wa m/z uli wocheperapo kuposa mtengo wa precursor ndipo kugawikana kwa MS/MS kumachitika pamalo omwewo a chitsanzo mu deta yomwe idakhazikitsidwa ndi precursor yomwe idachokera, ndi pomwe imaonedwa ngati fragment.
Kutengera malamulo awiri osavuta awa, timachotsa zidutswa zomwe zatchulidwa zomwe zili ndi ma m/z ochulukirapo kuposa m/z wa precursor wodziwika, komanso kutengera malo omwe precursor akuwonekera ndi chidutswa chomwe chatchulidwa. N'zothekanso kusankha mawonekedwe abwino omwe amapangidwa ndi zidutswa zambiri zomwe zimapangidwa mu MS1 ​​mode ngati precursors, zomwe zimapangitsa kuti pakhale ma MS/MS ochulukirapo. Pofuna kuchepetsa kuchuluka kwa deta kumeneku, ngati kufanana kwa NDP kwa ma spectra kupitirira 0.6, ndipo ali mu chromatogram "pcgroup" yolembedwa ndi CAMERA, tidzaziphatikiza. Pomaliza, timaphatikiza zotsatira zonse zinayi za CE zokhudzana ndi precursor ndi zidutswa mu composite spectrum yomaliza yodulidwa posankha peak yapamwamba kwambiri pakati pa ma candidate peak onse omwe ali ndi mtengo womwewo wa m/z pa mphamvu zosiyanasiyana za collision. Njira zotsatizana zokonzera zimachokera pa lingaliro la composite spectrum ndipo timaganizira mikhalidwe yosiyanasiyana ya CE yomwe imafunika kuti pakhale mwayi wogawa, chifukwa zidutswa zina zimatha kuzindikirika pogwiritsa ntchito mphamvu inayake ya collision.
RDPI (30) idagwiritsidwa ntchito kuwerengera kusinthasintha kwa mawonekedwe a kagayidwe kachakudya. Kusiyana kwa metabolic spectrum (Hj index) kumachokera ku kuchuluka kwa ma precursors a MS/MS pogwiritsa ntchito Shannon entropy ya kugawa kwa MS/MS frequency pogwiritsa ntchito equation yotsatirayi yomwe yafotokozedwa ndi Martínez et al. (8). Hj = −∑i = 1mPijlog2(Pij) pomwe Pij ikugwirizana ndi pafupipafupi ya i-th MS/MS mu chitsanzo cha j-th (j = 1, 2,…, m) (i = 1, 2, …, m) t).
Kufotokozera za kagayidwe kachakudya (Si index) kumatanthauzidwa ngati chizindikiritso cha MS/MS yoperekedwa poyerekeza ndi kuchuluka kwa zitsanzo zomwe zikuganiziridwa. Kufotokozera za MS/MS kumawerengedwa ngati Si = 1t (∑j = 1tPijPilog2PijPi)
Gwiritsani ntchito njira yotsatirayi kuti muyesere chizindikiro cha metabolome-specific δj cha chitsanzo chilichonse cha j, ndi avareji ya MS/MS specificity δj = ∑i = 1mPijSi
Ma spectra a MS/MS amalumikizidwa m'magulu awiriawiri, ndipo kufanana kumawerengedwa kutengera zigoli ziwirizo. Choyamba, pogwiritsa ntchito njira ya standard NDP (yomwe imadziwikanso kuti cosine correlation method), gwiritsani ntchito equation yotsatirayi kuti mupeze kufanana kwa gawo pakati pa ma spectra NDP = (∑iS1 & S2WS1, iWS2, i) 2∑iWS1, i2∑iWS2, i2 pomwe S1 ndi S2 Momwemonso, pa spectrum 1 ndi spectrum 2, komanso WS1, i ndi WS2, i imayimira kulemera kutengera mphamvu ya peak kuti kusiyana kwa i-th common peak pakati pa ma spectra awiriwa ndi kochepera 0.01 Da. Kulemera kumawerengedwa motere: W = [peak intensity] m [quality] n, m = 0.5, n = 2, monga momwe MassBank ikufotokozera.
Njira yachiwiri yowerengera inagwiritsidwa ntchito, yomwe inaphatikizapo kusanthula NL yogawana pakati pa MS/MS. Pachifukwa ichi, tinagwiritsa ntchito mndandanda wa 52 NL womwe umakumana nawo nthawi zambiri panthawi yogawa MS pamodzi, ndi NL yeniyeni (fayilo ya data S1) yomwe idalembedwa kale za MS/MS spectrum ya metabolites yachiwiri ya Nepenthes yofooka (9, 26). Pangani vekitala ya binary ya 1 ndi 0 pa MS/MS iliyonse, yogwirizana ndi yapano komanso yosakhalapo ya NL ina motsatana. Kutengera kufanana kwa mtunda wa Euclidean, chiŵerengero cha kufanana kwa NL chimawerengedwa pa awiriawiri aliwonse a binary NL vectors.
Kuti tichite ma clustering awiri, tinagwiritsa ntchito phukusi la R DiffCoEx, lomwe limachokera ku extension ya Weighted Gene Co-expression Analysis (WGCNA). Pogwiritsa ntchito NDP ndi NL scoring matrices a MS/MS spectra, tinagwiritsa ntchito DiffCoEx kuwerengera matrix yofananira. Binary clustering imachitika poika "cutreeDynamic" parameter kukhala njira = "hybrid", cutHeight = 0.9999, deepSplit = T, ndi minClusterSize = 10. R source code ya DiffCoEx idatsitsidwa kuchokera ku fayilo yowonjezera 1 ndi Tesson et al. (57); Phukusi la mapulogalamu a R WGCNA lofunikira lingapezeke mu https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA.
Kuti tichite kusanthula kwa ma netiweki a MS/MS, tinawerengera kulumikizana kwa ma spectral awiriawiri kutengera mitundu yofanana ya NDP ndi NL, ndipo tinagwiritsa ntchito pulogalamu ya Cytoscape kuti tiwone topology ya netiweki pogwiritsa ntchito kapangidwe kachilengedwe mu pulogalamu yowonjezera ya CyFilescape yFiles layout algorithm.
Gwiritsani ntchito R version 3.0.1 kuti mufufuze ziwerengero pa deta. Kufunika kwa ziwerengero kunayesedwa pogwiritsa ntchito njira ziwiri zowunikira kusiyana kwa kusiyana (ANOVA), kutsatiridwa ndi mayeso a Tukey a kusiyana kwakukulu (HSD) pambuyo pa hoc. Pofuna kusanthula kusiyana pakati pa chithandizo cha herbivorous ndi control, kugawa kwa mizere iwiri kwa magulu awiri a zitsanzo zomwe zili ndi kusiyana komweko kunafufuzidwa pogwiritsa ntchito mayeso a Student's t.
Kuti mudziwe zambiri zowonjezera pa nkhaniyi, chonde onani http://advances.sciencemag.org/cgi/content/full/6/24/eaaz0381/DC1
Iyi ndi nkhani yotseguka yogawidwa motsatira malamulo a Creative Commons Attribution-Non-Commercial License, yomwe imalola kugwiritsa ntchito, kufalitsa ndi kubwerezanso mu njira iliyonse, bola ngati kugwiritsidwa ntchito komaliza sikuli kopindulitsa pamalonda ndipo mfundo ndi yakuti ntchito yoyambirira ndi yolondola.
Dziwani: Tikukupemphani kuti mupereke imelo yanu kuti munthu amene mwamulimbikitsa patsamba lino adziwe kuti mukufuna kuti aone imeloyo ndipo si sipamu. Sitidzalemba ma adilesi aliwonse a imelo.
Funso ili limagwiritsidwa ntchito poyesa ngati ndinu mlendo ndikuletsa kutumiza sipamu yokha.
Chiphunzitso cha chidziwitso chimapereka ndalama yodziwika bwino yofananizira ma metabolomes apadera ndi kulosera malingaliro a chitetezo cha mayeso.
Chiphunzitso cha chidziwitso chimapereka ndalama yodziwika bwino yofananizira ma metabolomes apadera ndi kulosera malingaliro a chitetezo cha mayeso.
©2021 American Association for the Advancement of Science. maumwini onse ndi otetezedwa. AAAS ndi mnzake wa HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef ndi COUNTER. ScienceAdvances ISSN 2375-2548.