Ka'idojin kare tsirrai daban-daban suna ba da muhimmiyar jagorar ka'ida don bayyana yanayin metabolism na musamman na tsire-tsire, amma har yanzu ana gwada manyan hasashensu. A nan, mun yi amfani da nazarin tandem mass spectrometry (MS/MS) mara son kai don bincika metabolome na nau'ikan taba da aka rage daga tsire-tsire daban-daban zuwa yawan jama'a da nau'ikan da ke da alaƙa da juna, kuma mun sarrafa adadi mai yawa na ka'idojin fasalin taro bisa ga yanayin da ke cikin bayanin. Tsarin don gwada manyan hasashen ka'idojin tsaro mafi kyau (OD) da ka'idojin manufa mai motsi (MT). Bangaren bayanai na metabolomics na tsire-tsire ya yi daidai da ka'idar OD, amma ya saba wa babban hasashen ka'idar MT akan yanayin metabolism da herbivores ke haifarwa. Daga micro zuwa ma'aunin juyin halitta na macro, an gano siginar jasmonate a matsayin babban abin da ke tantance OD, yayin da siginar ethylene ta ba da daidaitaccen daidaitawa na martanin takamaiman herbivore wanda cibiyar sadarwa ta kwayoyin MS/MS ta bayyana.
Magungunan metabolite na musamman waɗanda ke da tsari daban-daban sune manyan masu shiga cikin daidaitawar tsirrai da muhalli, musamman wajen kare maƙiya (1). Bambancin yanayin metabolism na musamman da aka samu a cikin tsirrai ya ƙarfafa shekaru da yawa na zurfafa bincike kan fannoni da yawa na ayyukan muhalli, kuma ya samar da dogon jerin ka'idojin kare tsirrai, waɗanda sune ci gaban juyin halitta da muhalli na hulɗar tsirrai da kwari. Binciken gwaji yana ba da jagora mai mahimmanci (2). Duk da haka, waɗannan ka'idodin kare tsirrai ba su bi hanyar da aka saba amfani da ita ta hanyar tunani mai zurfi ba, wanda manyan hasashen suna kan matakin bincike ɗaya (3) kuma an gwada su ta hanyar gwaji don ci gaba da zagaye na gaba na ci gaban ka'ida (4). Iyakokin fasaha suna iyakance tattara bayanai zuwa takamaiman nau'ikan metabolism kuma suna ware cikakken bincike na metabolites na musamman, don haka hana kwatancen tsakanin rukuni waɗanda suke da mahimmanci don ci gaban ka'ida (5). Rashin cikakkun bayanai na metabolites da kuɗi iri ɗaya don kwatanta aikin sarrafa sararin metabolism tsakanin ƙungiyoyin shuka daban-daban yana hana balagar kimiyya na filin.
Sabbin ci gaba a fannin nazarin metabolomics na tandem mass spectrometry (MS/MS) na iya fayyace canje-canjen rayuwa a cikin da tsakanin nau'ikan wani tsari na musamman, kuma ana iya haɗa su da hanyoyin lissafi don ƙididdige kamanceceniya tsakanin waɗannan gaurayawan hadaddun abubuwa. Ilimin sunadarai na baya (5). Haɗin fasahohin zamani a cikin bincike da lissafi yana ba da tsarin da ake buƙata don gwaji na dogon lokaci na hasashen da yawa da ka'idojin muhalli da juyin halitta suka yi na bambancin rayuwa. Shannon (6) ya gabatar da ka'idar bayanai a karon farko a cikin labarinsa a cikin 1948, yana kafa harsashin nazarin lissafi na bayanai, wanda aka yi amfani da shi a fannoni da yawa banda aikace-aikacensa na asali. A cikin ilimin halittar jiki, an yi amfani da ka'idar bayanai cikin nasara don auna bayanai masu ra'ayin mazan jiya (7). A cikin binciken transcriptomics, ka'idar bayanai tana nazarin canje-canje gabaɗaya a cikin rubutun (8). A cikin binciken da ya gabata, mun yi amfani da tsarin kididdiga na ka'idar bayanai ga metabolomics don bayyana ƙwarewar metabolism na matakin nama a cikin tsire-tsire (9). A nan, mun haɗa tsarin aiki na tushen MS/MS tare da tsarin ƙididdiga na ka'idar bayanai, wanda aka siffanta shi da bambancin metabolism a cikin kuɗin gama gari, don kwatanta manyan hasashen ka'idar kare tsirrai na metabolome da masu cin ganyayyaki ke haifarwa.
Tsarin ka'idar kariyar tsirrai yawanci suna da haɗin kai kuma ana iya raba su zuwa rukuni biyu: waɗanda ke ƙoƙarin bayyana rarrabawar metabolites na musamman ga tsire-tsire bisa ga ayyukan kariya, kamar ingantaccen tsaro (OD) (10), manufa mai motsi (MT) (11)) Da kuma ka'idar bayyanar (12), yayin da wasu ke neman bayanin injiniya game da yadda canje-canje a cikin wadatar albarkatu ke shafar girman shuka da tarin metabolites na musamman, kamar carbon: hasashen daidaiton abinci mai gina jiki (13), hasashen ƙimar girma (14), da kuma hasashen daidaiton girma da bambance-bambance (15). Ka'idojin guda biyu suna cikin matakai daban-daban na bincike (4). Duk da haka, ka'idoji guda biyu da suka shafi ayyukan kariya a matakin aiki sun mamaye tattaunawar game da kariyar tsirrai da ta hanyar da za a iya amfani da ita: ka'idar OD, wacce ke ɗauka cewa tsire-tsire suna saka hannun jari a cikin kariyar sinadarai masu tsada kawai lokacin da ake buƙata, misali, lokacin da aka cinye su Lokacin da dabbar ciyawa ta kai hari, saboda haka, bisa ga yuwuwar hari a nan gaba, an sanya mahaɗin da ke da aikin kariya (10); Hasashe na MT ya nuna cewa babu wani ginshiƙi na canjin metabolite na alkibla, amma metabolite yana canzawa bazuwar, don haka yana haifar da yuwuwar Hana "maƙasudin motsi" na metabolism na kai hari ga masu cin ganyayyaki. A wata ma'anar, waɗannan ka'idoji guda biyu suna yin hasashen akasin haka game da sake fasalin metabolism wanda ke faruwa bayan harin masu cin ganyayyaki: alaƙar da ke tsakanin tarin metabolites tare da aikin kariya (OD) da canje-canje na metabolism marasa jagora (MT) (11).
Hasashe na OD da MT ba wai kawai sun ƙunshi canje-canjen da aka haifar a cikin metabolome ba, har ma da sakamakon muhalli da juyin halitta na tarin waɗannan metabolites, kamar farashin daidaitawa da fa'idodin waɗannan canje-canjen metabolism a cikin wani takamaiman yanayin muhalli (16). Duk da cewa duka hasashe sun fahimci aikin kariya na metabolites na musamman, waɗanda ƙila su yi tsada ko ba za su yi tsada ba, babban hasashen da ke bambanta hasashe na OD da MT yana cikin alkiblar canje-canjen metabolism da aka haifar. Hasashen ka'idar OD ya sami kulawa mafi girma zuwa yanzu. Waɗannan gwaje-gwajen sun haɗa da nazarin ayyukan kariya kai tsaye ko kai tsaye na kyallen takarda daban-daban na takamaiman mahadi a cikin gidajen kore da yanayin halitta, da kuma canje-canje a matakin ci gaban tsirrai (17-19). Duk da haka, zuwa yanzu, saboda rashin tsarin aiki da tsarin ƙididdiga don cikakken nazarin bambancin metabolism na duniya na kowace halitta, babban hasashen bambanci tsakanin ka'idoji biyu (wato, alkiblar canje-canjen metabolism) har yanzu ba a gwada shi ba. A nan, mun bayar da irin wannan bincike.
Ɗaya daga cikin muhimman halaye na metabolites na musamman ga tsire-tsire shine bambancin tsarinsu mai tsanani a kowane mataki daga tsire-tsire ɗaya, yawan jama'a zuwa nau'ikan iri ɗaya (20). Ana iya lura da canje-canje da yawa a cikin metabolites na musamman a sikelin yawan jama'a, yayin da bambance-bambancen inganci masu ƙarfi galibi ana kiyaye su a matakin nau'in (20). Saboda haka, bambancin metabolism na tsire-tsire shine babban ɓangaren bambancin aiki, yana nuna daidaitawa ga wurare daban-daban, musamman waɗancan wurare masu yuwuwar mamayewa daban-daban ta hanyar kwari na musamman da masu cin ganyayyaki na yau da kullun (21). Tun daga labarin Fraenkel (22) mai ban mamaki kan dalilan wanzuwar metabolites na musamman ga tsire-tsire, hulɗa da kwari daban-daban an ɗauke su a matsayin matsi mai mahimmanci na zaɓi, kuma ana tsammanin waɗannan hulɗar sun tsara tsire-tsire yayin juyin halitta. Hanyar metabolism (23). Bambancin nau'ikan halittu a cikin bambancin metabolites na musamman na iya nuna daidaiton ilimin halittar jiki da ke da alaƙa da kariyar tsire-tsire masu tsari da kuma waɗanda za a iya haifarwa daga dabarun herbivorous, saboda nau'ikan biyu galibi suna da alaƙa mara kyau da juna (24). Ko da yake yana da amfani a ci gaba da samun kyakkyawan kariya a kowane lokaci, canje-canjen rayuwa a kan kari da suka shafi tsaro a kan kari suna ba da fa'idodi bayyanannu wajen ba da damar shuke-shuke su ware albarkatu masu mahimmanci ga wasu jarin jiki (19, 24), da kuma guje wa buƙatar haɗuwa. Lalacewar haɗin gwiwa (25). Bugu da ƙari, waɗannan sake tsara ƙwayoyin halitta na musamman waɗanda ƙwayoyin kwari ke haifarwa na iya haifar da rarrabawa mai lalacewa a cikin jama'a (26), kuma suna iya nuna karatun kai tsaye na canje-canje na halitta masu yawa a cikin siginar jasmonic acid (JA), wanda za a iya kiyayewa a cikin jama'a. Siginar JA mai girma da ƙasa ciniki ne tsakanin kariya daga masu ciyawa da gasa da takamaiman nau'ikan halittu (27). Bugu da ƙari, hanyoyin ƙwayoyin halitta na metabolite na musamman za su fuskanci asara da canji cikin sauri yayin juyin halitta, wanda ke haifar da rarrabawar metabolism mara iyaka tsakanin nau'ikan halittu masu alaƙa (28). Waɗannan bambance-bambancen halittu za a iya kafa su cikin sauri don mayar da martani ga canza yanayin herbivore (29), wanda ke nufin cewa canjin al'ummomin herbivore babban abin da ke haifar da bambancin rayuwa.
A nan, mun magance matsalolin da ke tafe musamman. (I) Ta yaya ƙwarin da ke da ganye ke sake tsara tsarin metabolism na shuka? (Ii) Waɗanne manyan abubuwan da ke tattare da bayanai game da yanayin metabolism waɗanda za a iya ƙididdige su don gwada hasashen ka'idar tsaro ta dogon lokaci? (Iii) Ko za a sake tsara tsarin metabolism na shuka ta wata hanya ta musamman ga mai kai hari, idan haka ne, wace rawa hormone na shuka ke takawa wajen daidaita takamaiman martanin metabolism, kuma waɗanne metabolites ke ba da gudummawa ga takamaiman nau'in kariya? (Iv) Tunda hasashen da ka'idojin tsaro da yawa suka yi za a iya faɗaɗa su a duk matakan kyallen halitta, mun tambayi yadda martanin metabolism da aka haifar yake daidai daga kwatancen ciki zuwa kwatancen nau'ikan halittu daban-daban? Don haka, mun yi nazari kan tsarin sinadarin nicotine na taba a cikin ganyayyaki, wanda shuka ce ta muhalli mai wadataccen tsarin narkewar abinci, kuma tana da tasiri ga tsutsotsi na masu cin ganyayyaki guda biyu na asali, Lepidoptera Datura (Ms) (Mai tsananin ƙarfi, galibi ana cin ta). A kan Solanaceae da Spodoptera littoralis (Sl), tsutsotsin ganyen auduga wani nau'in "nau'in halitta", tare da tsire-tsire masu karɓar bakuncin Solanaceae da sauran masu karɓar sauran nau'ikan da iyalai Abincin shuka. Mun yi nazarin bakan metabolomics na MS/MS da kuma bayanan kididdiga na ka'idar bayanai don kwatanta ka'idojin OD da MT. Ƙirƙiri taswira na musamman don bayyana asalin mahimman metabolites. An faɗaɗa binciken zuwa ga al'ummar N. nasi da nau'ikan taba masu alaƙa don ƙarin nazarin bambancin da ke tsakanin siginar hormone na shuka da kuma shigar da OD.
Domin kama taswirar gabaɗaya game da ƙarfin jiki da tsarin metabolome na ganyen taba na herbivorous, mun yi amfani da wani bincike da tsarin lissafi da aka riga aka tsara don tattarawa da kuma rage yawan bayanai masu ƙuduri mai zurfi daga abubuwan da aka cire daga tsire-tsire (9). Wannan hanyar da ba ta bambanta ba (wanda ake kira MS/MS) na iya gina abubuwan da ba su da yawa, wanda za'a iya amfani da shi don duk nazarin matakin mahadi da aka bayyana a nan. Waɗannan abubuwan da aka cire daga tsirrai suna da nau'ikan iri daban-daban, waɗanda suka ƙunshi ɗaruruwa zuwa dubban metabolites (kimanin 500-1000-s/MS/MS a nan). A nan, muna la'akari da ƙarfin jiki a cikin tsarin ka'idar bayanai, kuma muna ƙididdige bambancin da ƙwarewar metabolome bisa ga Shannon entropy na rarraba mitar metabolism. Ta amfani da dabarar da aka aiwatar a baya (8), mun ƙididdige saitin alamomi waɗanda za a iya amfani da su don ƙididdige bambancin metabolome (alamar Hj), ƙwarewar bayanin martaba na metabolism (alamar δj) da takamaiman yanayin metabolism na metabolite guda ɗaya (alamar Si). Bugu da ƙari, mun yi amfani da Ma'aunin Nisa na Plastik na Dangantaka (RDPI) don auna ƙarfin metabolome na masu cin ganyayyaki (Hoto na 1A) (30). A cikin wannan tsarin ƙididdiga, muna ɗaukar bakan MS/MS a matsayin sashin bayanai na asali, kuma muna sarrafa yawan MS/MS a cikin taswirar rarraba mita, sannan mu yi amfani da Shannon entropy don kimanta bambancin metabolome daga gare ta. Ana auna ƙwarewar metabolome ta hanyar matsakaicin takamaiman bakan MS/MS guda ɗaya. Saboda haka, ƙaruwar yawan wasu azuzuwan MS/MS bayan haɓakar herbivore an canza shi zuwa inducibility na spectral, RDPI da ƙwarewa, wato, ƙaruwar ma'aunin δj, saboda ana samar da ƙarin metabolites na musamman kuma ana samar da babban ma'aunin Si. Rage ma'aunin bambancin Hj yana nuna cewa ko dai adadin MS/MS da aka samar ya ragu, ko kuma rarraba mitar bayanin martaba yana canzawa a cikin alkibla mara daidaito, yayin da yake rage rashin tabbas gabaɗaya. Ta hanyar lissafin ma'aunin Si, yana yiwuwa a haskaka waɗanne MS/MS ne wasu masu cin ganyayyaki ke haifarwa, akasin haka, wanda MS/MS ba ya amsawa ga induction, wanda shine babban alamar bambance hasashen MT da OD.
(A) Bayanan kididdiga da aka yi amfani da su don haɓaka bayanai na MS/MS (H1 zuwa Hx), bambancin (Hj index), ƙwarewa (δj index) da takamaiman metabolite (Si index). Ƙara yawan matakin ƙwarewa (δj) yana nuna cewa, a matsakaici, za a samar da ƙarin metabolites na musamman na ganye, yayin da raguwar bambancin (Hj) yana nuna raguwar samar da metabolites ko rarraba metabolites mara daidaito a cikin taswirar rarrabawa. Ƙimar Si tana tantance ko metabolite ɗin ya keɓance ga wani yanayi da aka bayar (a nan, herbivorous) ko kuma akasin haka ana kiyaye shi a matakin ɗaya. (B) Tsarin ra'ayi na hasashen ka'idar tsaro ta amfani da ma'aunin ka'idar bayanai. Ka'idar OD ta annabta cewa harin herbivor zai ƙara metabolites na tsaro, ta haka yana ƙara δj. A lokaci guda, Hj yana raguwa saboda an sake tsara bayanin martaba zuwa ga raguwar rashin tabbas na bayanan metabolism. Ka'idar MT ta annabta cewa harin masu cin ganyayyaki zai haifar da canje-canje marasa alkibla a cikin metabolism, ta haka yana ƙara Hj a matsayin alamar ƙaruwar rashin tabbas game da bayanai na rayuwa da kuma haifar da rarraba Si bazuwar. Mun kuma gabatar da wani tsari mai gauraya, mafi kyawun MT, inda wasu metabolites tare da ƙimar kariya mafi girma za su ƙara musamman (ƙimar Si mai girma), yayin da wasu kuma ke nuna martani bazuwar (ƙananan ƙimar Si).
Ta amfani da bayanin ka'idar bayanai, mun fassara ka'idar OD don annabta cewa canje-canje na metabolite na musamman da ganye ke haifarwa a cikin yanayin da ba a haifar da shi ba zai haifar da (i) ƙaruwa a cikin takamaiman metabolism (Si index) yana haifar da takamaiman metabonomic (δj index). Ƙarawa na) wasu ƙungiyoyi na metabolite na musamman tare da ƙimar kariya mafi girma, da (ii) raguwar bambancin metabolome (Hj index) saboda canjin rarraba mitar metabolism zuwa ƙarin rarraba jiki na leptin. A matakin metabolite guda ɗaya, ana sa ran rarraba Si da aka tsara, inda metabolite zai ƙara ƙimar Si bisa ga ƙimar kariyarsa (Hoto na 1B). Tare da wannan layin, mun bayyana ka'idar MT don annabta cewa motsawa zai haifar da (i) canje-canje marasa alkibla a cikin metabolites wanda ke haifar da raguwa a cikin δj index, da (ii) ƙaruwa a cikin Hj index saboda ƙaruwar rashin tabbas na metabolism. Ko kuma bazuwar, wanda entropy na Shannon zai iya ƙididdigewa a cikin nau'in bambancin gabaɗaya. Dangane da abun da ke cikin metabolism, ka'idar MT za ta annabta rarraba bazuwar Si. Idan aka yi la'akari da cewa wasu metabolites suna ƙarƙashin takamaiman yanayi a ƙarƙashin takamaiman yanayi, kuma wasu yanayi ba sa ƙarƙashin takamaiman yanayi, kuma ƙimar kariyarsu ta dogara da muhalli, mun kuma gabatar da tsarin kariya mai gauraya, inda aka rarraba δj da Hj a cikin biyu tare da Si Increase a kowane bangare, wasu ƙungiyoyin metabolites ne kawai, waɗanda ke da ƙimar kariya mafi girma, za su ƙara Si musamman, yayin da wasu za su sami rarrabawa bazuwar (Hoto na 1B).
Domin gwada hasashen ka'idar kariya da aka sake fasalta a kan ma'aunin bayanin ka'idar bayanai, mun ɗaga ƙwarƙwarar ƙwararrun masu shuka ciyawa (Ms) ko kuma janar (Sl) a kan ganyen Nepenthes pallens (Hoto na 2A). Ta amfani da nazarin MS/MS, mun samo 599 na MS/MS marasa yawa (fayil ɗin bayanai S1) daga abubuwan da aka cire daga methanol na kyallen ganye da aka tattara bayan ciyar da ƙwarƙwata. Ta amfani da ma'aunin RDPI, Hj, da δj don ganin sake saita abubuwan da ke cikin bayanai a cikin fayilolin daidaitawar MS/MS yana bayyana alamu masu ban sha'awa (Hoto na 2B). Babban yanayin shine, kamar yadda mai bayanin bayanin ya bayyana, yayin da ƙwarƙwata ke ci gaba da cin ganye, matakin duk sake tsara tsarin rayuwa yana ƙaruwa akan lokaci: awanni 72 bayan cin ciyawar, RDPI yana ƙaruwa sosai. Idan aka kwatanta da ikon sarrafawa mara lalacewa, Hj ya ragu sosai, wanda ya faru ne saboda ƙaruwar matakin ƙwarewa na bayanin rayuwa, wanda aka ƙididdige shi ta hanyar ma'aunin δj. Wannan yanayin da ake gani ya yi daidai da hasashen ka'idar OD, amma bai yi daidai da manyan hasashen ka'idar MT ba, wanda ya yi imanin cewa canje-canje bazuwar (marasa alkibla) a matakan metabolite ana amfani da su azaman ɓoye kariya (Hoto na 1B). Duk da cewa yawan fitar da ruwa daga baki (OS) da kuma yadda ake ciyar da waɗannan masu cin ganyayyaki biyu sun bambanta, ciyarwarsu kai tsaye ta haifar da irin waɗannan canje-canje a alkiblar Hj da δj a lokacin girbi na awanni 24 da awanni 72. Bambancin kawai ya faru ne a awanni 72 na RDPI. Idan aka kwatanta da wanda ciyarwar Ms ta haifar, yawan narkewar abinci da ciyarwar Sl ta haifar ya fi girma.
(A) Tsarin gwaji: Ana ciyar da alade (S1) ko ƙwararrun (Ms) masu cin ganyayyaki da ganyen da aka cire gishiri daga tsire-tsire masu tukwane, yayin da ga ciyawar da aka kwaikwayi, ana amfani da tsarin Ms (W + OSMs) don magance raunin wurin da ganyen ke kasancewa. Ƙwayoyin S1 (W + OSSl) ko ruwa (W + W). Kulawa (C) ganye ne mara lalacewa. (B) Inducibility (idan aka kwatanta da jadawalin sarrafawa), bambancin (Hj index) da kuma specialization (δj index) index da aka ƙididdige don taswirar metabolite ta musamman (599 MS/MS; fayil ɗin bayanai S1). Alamun tauraro suna nuna bambance-bambance masu mahimmanci tsakanin ciyar da ciyawa kai tsaye da ƙungiyar kulawa (gwajin t-ɗalibi tare da gwajin t-gwajin da aka haɗa, *P<0.05 da ***P<0.001). ns, ba mahimmanci ba ne. (C) Ma'aunin ƙudurin lokaci na babban (akwatin shuɗi, amino acid, acid na halitta da sukari; fayil ɗin bayanai S2) da kuma takamaiman nau'in metabolite (akwatin ja 443 MS/MS; fayil ɗin bayanai S1) bayan maganin herbivory da aka kwaikwayi. Ramin launi yana nufin tazara ta amincewa ta 95%. Alamar ta nuna babban bambanci tsakanin magani da sarrafawa [binciken bambancin quadratic (ANOVA), sai kuma babban bambanci na Tukey (HSD) don kwatancen da yawa bayan hoc, *P<0.05, **P<0.01 Da *** P <0.001]. (D) Ƙwarewa a kan taswirar watsawa da bayanan metabolite na musamman (samfurori masu maimaitawa tare da jiyya daban-daban).
Domin bincika ko gyaran da herbivore ke haifarwa a matakin metabolome yana nuna canje-canje a matakin metabolites na mutum ɗaya, da farko mun mayar da hankali kan metabolites da aka yi nazari a kansu a cikin ganyen Nepenthes pallens tare da tabbataccen juriya ga herbivore. Phenolic amides sune hydroxycinnamamide-polyamine conjugates waɗanda ke taruwa a lokacin tsarin herbivore na kwari kuma an san su da rage aikin kwari (32). Mun bincika abubuwan da suka fara na MS/MS da suka dace kuma muka zana lanƙwasa na tarawar motsi (Hoto na S1). Abin mamaki, abubuwan da aka samo daga phenol waɗanda ba su da hannu kai tsaye a cikin kariya daga herbivores, kamar chlorogenic acid (CGA) da rutin, ba su da ƙarfi bayan herbivory. Akasin haka, herbivores na iya sa phenol amides ya yi ƙarfi sosai. Ci gaba da ciyar da herbivores guda biyu ya haifar da kusan irin wannan yanayin motsa jiki na phenolamides, kuma wannan tsari ya bayyana musamman ga haɗin phenolamides na de novo. Za a lura da irin wannan lamari yayin binciken hanyar 17-hydroxygeranyl nonanediol diterpene glycosides (17-HGL-DTGs), wanda ke samar da adadi mai yawa na diterpenes masu aiki da ingantaccen aikin hana ciyawa (33), wanda daga cikinsu Ms Feeding with Sl ya haifar da irin wannan bayanin bayyanar (Hoto na S1)).
Rashin da zai iya faruwa a gwaje-gwajen ciyar da tsirrai kai tsaye shine bambancin yawan cin ganye da lokacin ciyar da tsirrai, wanda hakan ke sa ya zama da wahala a kawar da tasirin da ganye ke yi wa ganyen da raunuka da ciyawa ke haifarwa. Domin mu magance takamaiman martanin da ganyen ke haifarwa, mun yi kwaikwayon ciyar da tsutsotsi na Ms da Sl ta hanyar amfani da OS (OSM da OSS1) nan da nan zuwa ga daidaitattun wurare na huda ganye. Wannan hanyar ana kiranta maganin W + OS, kuma tana daidaita motsawar ta hanyar daidaita lokacin fara amsawar da ganyen ke haifarwa ba tare da haifar da tasirin rikice-rikice na bambance-bambance a cikin ƙimar ko adadin asarar nama ba (Hoto na 2A) (34). Ta amfani da hanyar bincike da ƙididdige MS/MS, mun samo 443 MS/MS spectra (fayil ɗin bayanai S1), wanda ya yi karo da spectra da aka riga aka tattara daga gwaje-gwajen ciyar da kai tsaye. Nazarin ka'idar bayanai na wannan saitin bayanai na MS/MS ya nuna cewa sake tsara metabolomes na ganye ta hanyar kwaikwayon herbivores ya nuna abubuwan da ke haifar da OS na musamman (Hoto na 2C). Musamman ma, idan aka kwatanta da maganin OSS1, OSM ya haifar da haɓaka ƙwarewar metabolome a cikin awanni 4. Ya kamata a lura cewa idan aka kwatanta da saitin bayanan gwaji na ciyar da herbivore kai tsaye, ana iya ganin motsin metabolism a cikin sarari mai girma biyu ta amfani da Hj da δj a matsayin daidaitawa da kuma alkiblar ƙwarewar metabolome a cikin martani ga maganin herbivore da aka kwaikwayi akan lokaci Ƙara daidaito (Hoto na 2D). A lokaci guda, mun ƙididdige abubuwan da ke cikin amino acid, acid na halitta da sukari (fayil ɗin bayanai S2) don bincika ko wannan ƙaruwar da aka yi niyya a cikin ƙwarewar metabolome ya faru ne saboda sake tsara metabolism na tsakiya na carbon a cikin martani ga herbivore da aka kwaikwayi (Hoto na S2). Don ƙarin bayani game da wannan tsarin, mun ƙara sa ido kan tasirin taruwar metabolism na hanyoyin phenolamide da 17-HGL-DTG da aka tattauna a baya. An canza tsarin OS-takamaiman shigar da herbivore zuwa tsarin sake fasalin daban-daban a cikin metabolism na phenolamide (Hoto na S3). OSS1 ne ke haifar da phenolic amides waɗanda ke ɗauke da coumarin da caffeoyl, yayin da OSMs ke haifar da takamaiman shigar da ferulyl conjugates. Don hanyar 17-HGL-DTG, an gano bambancin shigar OS ta hanyar malonylation da samfuran dimalonylation na ƙasa (Hoto na S3).
Na gaba, mun yi nazarin tsarin da OS ke haifar da tsarin aiki ta amfani da saitin bayanai na microarray na lokaci-lokaci, wanda ke kwaikwayon amfani da OSMs don magance ganyen ganyen rosette a cikin masu cin ganyayyaki. Tsarin samfurin ya yi daidai da tsarin da aka yi amfani da shi a cikin wannan binciken metabolomics (35). Idan aka kwatanta da sake tsara metabolome wanda ke ƙaruwa da tsarin metabolism musamman akan lokaci, mun lura da fashewar rubutun wucin gadi a cikin ganyayyaki da Ms ta haifar, inda inducibility na transcriptionome (RDPI) da ƙwarewa (δj) suke a 1 Akwai ƙaruwa mai yawa a cikin sa'o'i, kuma bambancin (Hj) a wannan lokacin, an rage yawan bayyanar BMP1 sosai, sannan aka biyo baya da sassauta ƙwarewar transcriptome (Hoto na S4). Iyalan kwayoyin halitta na metabolism (kamar P450, glycosyltransferase, da BAHD acyltransferase) suna shiga cikin tsarin haɗa metabolites na musamman daga sassan tsarin da aka samo daga metabolism na farko, bin samfurin farko na ƙwarewa. A matsayin wani bincike, an yi nazarin hanyar phenylalanine. Binciken ya tabbatar da cewa manyan kwayoyin halitta a cikin metabolism na phenolamide suna haifar da OS sosai a cikin masu cin ganyayyaki idan aka kwatanta da tsire-tsire marasa sha'awa, kuma suna da alaƙa sosai a cikin tsarin bayyanar su. Factor na kwafi na MYB8 da kwayoyin halittar tsarin PAL1, PAL2, C4H da 4CL a cikin wannan hanyar sun nuna farkon fara kwafi. Acyltransferases waɗanda ke taka rawa a cikin haɗuwa ta ƙarshe na phenolamide, kamar AT1, DH29, da CV86, suna nuna tsarin haɓakawa mai tsawo (Hoto na S4). Abubuwan da aka lura a sama sun nuna cewa farkon farawar ƙwarewa ta transcriptome da haɓaka ƙwarewa ta metabolomics daga baya yanayi ne mai haɗin gwiwa, wanda wataƙila ya faru ne saboda tsarin daidaitawa wanda ke fara amsawa mai ƙarfi na tsaro.
Sake fasalin siginar hormone na shuka yana aiki azaman layin tsari wanda ke haɗa bayanan ganye don sake tsara yanayin tsirrai. Bayan kwaikwayon herbivore, mun auna tarin ƙarfin hormones na manyan nau'ikan shuke-shuke kuma mun hango haɗin gwiwa tsakanin su [Pearson correlation coefficient (PCC)> 0.4] (Hoto na 3A). Kamar yadda aka zata, hormones na shuka da ke da alaƙa da biosynthesis suna da alaƙa a cikin hanyar sadarwa ta haɗin hormone na shuka. Bugu da ƙari, an tsara takamaiman metabolism (Si index) zuwa wannan hanyar sadarwa don haskaka hormones na shuka da magunguna daban-daban suka haifar. An zana manyan fannoni biyu na takamaiman amsawar herbivore: ɗaya yana cikin rukunin JA, inda JA (nau'in JA-Ile mai aiki a fannin halitta) da sauran abubuwan da aka samo daga JA suna nuna mafi girman maki Si; ɗayan kuma shine ethylene (ET). Gibberellin ya nuna matsakaicin ƙaruwa a cikin takamaiman herbivore, yayin da sauran hormones na shuka, kamar cytokinin, auxin, da abscisic acid, suna da ƙarancin takamaiman induction ga herbivore. Idan aka kwatanta da amfani da W + W kaɗai, ƙaruwar ƙimar kololuwar abubuwan da aka samo daga JA ta hanyar aikace-aikacen OS (W + OS) za a iya canza ta zuwa wata alama mai ƙarfi ta musamman ta JAs. Ba zato ba tsammani, an san cewa OSM da OSS1 tare da abubuwan da ke haifar da ƙwanƙwasa daban-daban suna haifar da tarin JA da JA-Ile iri ɗaya. Sabanin OSS1, OSMs ne ke haifar da OSM musamman kuma mai ƙarfi, yayin da OSS1 ba ya ƙara yawan amsawar raunukan basal (Hoto na 3B).
(A) Binciken hanyar sadarwa ta hanyar amfani da PCC bisa ga lissafin PCC na kwaikwayon motsin tara hormones na shuka da herbivore ke haifarwa. Layin yana wakiltar hormone ɗaya na shuka, kuma girman layin yana wakiltar ma'aunin Si wanda ya kebanta da hormone na shuka tsakanin jiyya. (B) Tarin JA, JA-Ile da ET a cikin ganye wanda ya haifar da jiyya daban-daban da aka nuna ta launuka daban-daban: apricot, W + OSM; shuɗi, W + OSSl; baƙi, W + W; launin toka, C (iko). Alamun tauraro suna nuna bambance-bambance masu mahimmanci tsakanin magani da sarrafawa (ANOVA mai hanyoyi biyu sannan Tukey HSD ya biyo bayan kwatancen da yawa, *** P <0.001). Binciken ka'idar bayanai na (C)697 MS/MS (fayil ɗin bayanai S1) a cikin biosynthesis na JA da raunin fahimtar bakan (irAOC da irCOI1) da (D)585 MS/MS (fayil ɗin bayanai S1) a cikin ETR1 tare da raunin siginar ET Magungunan herbivore guda biyu da aka kwaikwayi sun haifar da layukan shuka da tsire-tsire masu sarrafa abin hawa mara komai (EV). Alamun tauraro suna nuna bambance-bambance masu mahimmanci tsakanin maganin W+OS da kuma kulawar da ba ta lalace ba (ANOVA mai hanyoyi biyu sannan sai Tukey HSD ya biyo bayan kwatancen da yawa, *P<0.05, **P<0.01 da ***P<0.001). (E) Jadawalin da aka watsa na adawa da ƙwarewa. Launuka suna wakiltar nau'ikan nau'ikan da aka gyara ta hanyar kwayoyin halitta; alamomin suna wakiltar hanyoyin magani daban-daban: alwatika, W + OSS1; murabba'i mai kusurwa huɗu, W + OSM; da'ira C
Na gaba, muna amfani da nau'in Nepenthes da aka gyara ta hanyar halitta (irCOI1 da sETR1) a cikin manyan matakan JA da ET biosynthesis (irAOC da irACO) da fahimta (irCOI1 da sETR1) don nazarin metabolism na waɗannan hormones guda biyu na shuka akan masu cin ganyayyaki. Gudunmawar da aka samu ta hanyar sake tsara shirye-shirye. Daidai da gwaje-gwajen da suka gabata, mun tabbatar da haifar da herbivore-OS a cikin tsire-tsire masu ɗaukar hoto mara komai (EV) (Hoto na 3, C zuwa D) da raguwar gabaɗaya a cikin ma'aunin Hj da OSM ya haifar, yayin da ma'aunin δj ya ƙaru. Amsar ta fi bayyana fiye da martanin da OSS1 ya haifar. Jadawalin layi biyu ta amfani da Hj da δj a matsayin daidaitawa yana nuna takamaiman ragewa (Hoto na 3E). Yanayin da ya fi bayyana shine a cikin nau'ikan da ba su da siginar JA, bambancin metabolome da canje-canje na musamman da masu cin ganyayyaki ke haifarwa kusan an kawar da su gaba ɗaya (Hoto na 3C). Sabanin haka, fahimtar ET a cikin tsire-tsire na sSETR1, kodayake tasirin gabaɗaya akan canje-canje a cikin metabolism na ganye yana ƙasa da na siginar JA, yana rage bambancin da ke cikin ma'aunin Hj da δj tsakanin abubuwan motsa jiki na OSM da OSS1 (Hoto na 3D da Hoto na S5). . Wannan yana nuna cewa ban da aikin babban aikin transduction na siginar JA, transduction na siginar ET kuma yana aiki azaman daidaita martanin metabolism na musamman na nau'in na masu herbivores. Daidai da wannan aikin gyarawa mai kyau, babu wani canji a cikin haɗin metabolism gabaɗaya a cikin tsire-tsire na sSETR1. A gefe guda kuma, idan aka kwatanta da tsire-tsire na sSETR1, tsire-tsire na irACO sun haifar da irin wannan girman canje-canje na metabolism wanda masu herbivores ke haifarwa, amma sun nuna maki Hj da δj daban-daban tsakanin ƙalubalen OSM da OSS1 (Hoto na S5).
Domin gano ƙwayoyin halitta na musamman waɗanda ke da muhimmiyar gudummawa ga amsawar nau'in ƙwayoyin halitta na masu cin ganyayyaki da kuma daidaita yadda suke samar da su ta hanyar siginar ET, mun yi amfani da hanyar MS/MS da aka ƙirƙiro a baya. Wannan hanyar ta dogara ne akan hanyar haɗa nau'i biyu don sake gano dangin metabolism daga gutsuttsuran MS/MS [samfurin digo mai tsari (NDP)] da maki kamanceceniya bisa ga asarar tsaka tsaki (NL). Saitin bayanai na MS/MS da aka gina ta hanyar nazarin layukan transgenic na ET ya samar da MS/MS 585 (fayil ɗin bayanai S1), wanda aka warware ta hanyar rarraba su zuwa manyan sassan MS/MS guda bakwai (M) (Hoto na 4A). Wasu daga cikin waɗannan sassan suna cike da ƙwayoyin halitta na musamman da aka riga aka siffanta: misali, M1, M2, M3, M4 da M7 suna da wadataccen nau'ikan phenol derivatives (M1), flavonoid glycosides (M2), acyl sugars (M3 Da M4), da 17-HGL-DTG (M7). Bugu da ƙari, ana ƙididdige takamaiman bayanai na metabolism (ma'aunin Si) na metabolite guda ɗaya a cikin kowane module, kuma ana iya ganin rarraba Si a hankali. A takaice, spectra na MS/MS da ke nuna babban herbivory da takamaiman genotype an siffanta su da babban ƙimar Si, kuma ƙididdigar kurtosis tana nuna rarrabar fur a kusurwar wutsiya ta dama. An gano wani irin wannan rarrabawar colloid mai laushi a cikin M1, inda phenol amide ya nuna mafi girman rabon Si (Hoto na 4B). An ambata a baya 17-HGL-DTG na herbivorous inducible a cikin M7 ya nuna matsakaicin maki Si, yana nuna matsakaicin matakin daidaitawa tsakanin nau'ikan OS guda biyu. Sabanin haka, yawancin metabolites na musamman da aka samar a cikin tsari, kamar rutin, CGA, da acyl sugars, suna cikin mafi ƙarancin maki Si. Domin a bincika sarkakiyar tsari da rarraba Si tsakanin metabolites na musamman, an gina hanyar sadarwa ta kwayoyin halitta don kowane module (Hoto na 4B). Wani muhimmin hasashe na ka'idar OD (wanda aka taƙaita a Hoto na 1B) shine cewa sake tsara metabolites na musamman bayan herbivory zai haifar da canje-canje na hanya ɗaya a cikin metabolites tare da ƙimar kariya mai girma, musamman ta hanyar ƙara takamaiman su (sabanin rarrabawa bazuwar) Yanayin kariya metabolite wanda ka'idar MT ta annabta. Yawancin abubuwan da aka samo daga phenol da aka tara a cikin M1 suna da alaƙa da raguwar aikin kwari (32). Lokacin da muke kwatanta ƙimar Si a cikin metabolites na M1 tsakanin ganyen da aka haifar da ganyen da aka haɗa da ganyen da ke sarrafa EV a cikin awanni 24, mun lura cewa takamaiman metabolism na metabolites da yawa bayan kwari masu cin ganyayyaki yana da babban ci gaba mai girma (Hoto na 4C). An gano takamaiman ƙaruwar ƙimar Si ne kawai a cikin phenolamides na kariya, amma ba a gano ƙaruwar ƙimar Si a cikin wasu phenols da metabolites da ba a san su ba waɗanda ke rayuwa tare da wannan tsarin. Wannan samfurin musamman ne, wanda ke da alaƙa da ka'idar OD. Babban hasashen canje-canje na metabolism da herbivory ke haifarwa suna da daidaito. Domin gwada ko wannan takamaiman yanayin phenolamide ya samo asali ne ta hanyar ET na musamman na OS, mun tsara ma'aunin metabolite Si kuma muka haifar da bambancin ƙimar magana tsakanin OSM da OSS1 a cikin nau'ikan genotypes na EV da sETR1 (Hoto na 4D). A cikin sETR1, bambancin da phenamide ya haifar tsakanin OSM da OSS1 ya ragu sosai. An kuma yi amfani da hanyar haɗa abubuwa biyu ga bayanan MS/MS da aka tattara a cikin nau'ikan da ba su da isasshen JA don gano manyan sassan MS/MS da suka shafi ƙwarewar metabolism ta JA (Hoto na S6).
(A) Sakamakon tarin MS/MS 585 bisa ga guntu da aka raba (kamanni na NDP) da asarar da aka raba (kamanni na NL) yana haifar da tsarin (M) ya yi daidai da dangin mahaɗin da aka sani, ko kuma ta hanyar abubuwan da ba a sani ba ko kuma waɗanda ba a metabolized ba. Kusa da kowane tsarin, an nuna rarrabawar metabolite (MS/MS) ta musamman (Si). (B) Cibiyar sadarwa ta kwayoyin halitta mai tsari: Nodes suna wakiltar MS/MS da gefuna, maki NDP (ja) da NL (shuɗi) MS/MS (yankewa,> 0.6). Ma'aunin takamaiman metabolite mai tsari (Si) wanda aka yi masa launi bisa tsarin (hagu) kuma an tsara shi zuwa hanyar sadarwa ta kwayoyin halitta (dama). (C) Tsarin M1 na shukar EV a cikin yanayin da aka haɗa (iko) da yanayin da aka haifar (mai kwaikwayon herbivore) a cikin awanni 24: zane-zanen hanyar sadarwa ta kwayoyin halitta (Ƙimar Si shine girman node, an nuna phenolamide mai kariya da shuɗi). (D) Zane-zanen hanyar sadarwa ta kwayoyin halitta ta M1 na layin bakan sETR1 tare da rashin fahimtar EV da ET: mahaɗin phenolic da aka wakilta ta hanyar koren da'irar kusurwoyi, da kuma babban bambanci (ƙimar P) tsakanin jiyya na W + OSM da W + OSS1 a matsayin girman 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 a cikin wolfberry Son; Nick. O-AS, O-acyl sugar.
Mun ƙara faɗaɗa binciken daga nau'in halittar Nepenthes guda ɗaya da aka rage zuwa ga al'ummomin halitta, inda aka riga aka bayyana canje-canje masu ƙarfi a cikin matakan JA na herbivorous da takamaiman matakan metabolite a cikin al'ummomin halitta (26). Yi amfani da wannan saitin bayanai don rufe ƙwayoyin cuta 43. Waɗannan ƙwayoyin cuta sun ƙunshi nau'ikan tsire-tsire 123 daga N. pallens. An ɗauko waɗannan tsire-tsire daga tsaba da aka tattara a cikin wurare daban-daban na asali a Utah, Nevada, Arizona, da California (Hoto na S7), mun ƙididdige bambancin metabolome (ana kiransa matakin yawan jama'a) bambancin β) da ƙwarewar da OSM ta haifar. Daidai da binciken da ya gabata, mun lura da sauye-sauye iri-iri na metabolism tare da gatari na Hj da δj, yana nuna cewa ƙwayoyin cuta suna da bambance-bambance masu mahimmanci a cikin ƙarfin amsawar metabolism ga masu herbivores (Hoto na S7). Wannan ƙungiyar tana tunawa da abubuwan da aka lura a baya game da kewayon canje-canjen JAs masu ƙarfi da masu herbivores ke haifarwa, kuma ta ci gaba da kasancewa mai girma a cikin al'umma ɗaya (26, 36). Ta hanyar amfani da JA da JA-Ile don gwada alaƙar matakin gabaɗaya tsakanin Hj da δj, mun gano cewa akwai muhimmiyar alaƙa mai kyau tsakanin JA da bambancin metabolome β da ma'aunin ƙwarewa (Hoto na S7). Wannan yana nuna cewa bambancin da herbivores ke haifarwa a cikin shigar JAs da aka gano a matakin jama'a na iya zama saboda manyan bambance-bambancen metabolism da ke faruwa ta hanyar zaɓi daga masu cin ganyayyaki.
Nazarin da aka yi a baya ya nuna cewa nau'ikan taba sun bambanta sosai a nau'in da kuma dogaro da juna kan kariyar metabolism da aka haifar da kuma ta hanyar tsari. Ana kyautata zaton cewa waɗannan canje-canje a cikin ikon watsa siginar hana ciyawa da kariya ana sarrafa su ne ta hanyar matsin lamba na yawan kwari, zagayowar rayuwar shuka, da kuma farashin samar da kariya a yankin da wani nau'in ya tsiro. Mun yi nazarin daidaiton sake fasalin metabolome na ganye wanda masu shuka na nau'ikan Nicotiana guda shida suka haifar a Arewacin Amurka da Kudancin Amurka. Waɗannan nau'ikan suna da alaƙa da Nepenthes Arewacin Amurka, wato Nicolas Bociflo. La, N. nicotinis, Nicotiana n. ciyawa mai rauni, Nicotiana tabacum, taba mai layi, taba (Nicotiana spegazzinii) da taba ganyen taba (Nicotiana obtusifolia) (Hoto na 5A) (37). Shida daga cikin waɗannan nau'ikan, gami da nau'ikan N. don Allah, tsire-tsire ne na shekara-shekara na petunia clade, kuma obtusifolia N. sune tsire-tsire na shekara-shekara na 'yar'uwar clade Trigonophyllae (38). Daga baya, an yi amfani da W + W, W + OSM da W + OSS1 a kan waɗannan nau'ikan guda bakwai don nazarin sake fasalin yanayin abinci na ciyar da kwari a matakin nau'in.
(A) Itacen bootstrap phylogenetic bisa ga mafi girman yuwuwar [don haɗakar glutamine na nukiliya (38)] da kuma rarrabawar yanki na nau'ikan Nicotiana guda bakwai masu alaƙa da juna (launuka daban-daban) (37). (B) Tsarin watsawa na bambancin musamman don bayanan metabolism na nau'ikan Nicotiana guda bakwai (939 MS/MS; fayil ɗin bayanai S1). A matakin nau'ikan, bambancin metabolome yana da alaƙa mara kyau da matakin ƙwarewa. An nuna nazarin alaƙar matakin nau'ikan tsakanin bambancin metabolism da ƙwarewa da tarin JA a cikin Hoto na 2. S9. Launi, nau'ikan daban-daban; alwatika, W + OSS1; murabba'i mai kusurwa huɗu, W + OSM; (C) An tsara yanayin Nicotiana JA da JA-Ile bisa ga girman motsin OS (kwatancen ANOVA mai hanyoyi biyu da Tukey HSD bayan kwatancen sau da yawa, * P <0.05, ** P <0.01 da * ** Don kwatanta W + OS da W + W, P <0.001). Tsarin akwati na (D) bambancin da (E) ƙwarewa ga kowane nau'in bayan kwaikwayon ganye da methyl JA (MeJA). Alamar ta nuna babban bambanci tsakanin W + OS da W + W ko lanolin da W (Lan + W) ko Lan da MeJA (Lan + MeJa) da kuma ikon sarrafa Lan (biyu-hanyoyi na bambance-bambance, sai kuma kwatancen Tukey na HSD bayan hoc da yawa, *P<0.05, **P<0.01 da ***P<0.001).
Ta amfani da hanyar rukuni biyu, mun gano kayayyaki 9 na 939 MS/MS (fayil ɗin bayanai S1). Tsarin MS/MS wanda aka sake tsara shi ta hanyoyi daban-daban ya bambanta sosai tsakanin kayayyaki daban-daban tsakanin nau'ikan (Hoto na S8). Ganin Hj (wanda aka ambata a nan a matsayin γ-bambancin nau'ikan) da δj ya nuna cewa nau'ikan daban-daban suna taruwa zuwa ƙungiyoyi daban-daban a cikin sararin metabolism, inda rarrabuwar matakin nau'ikan yawanci ya fi bayyana fiye da motsawa. Banda N. linear da N. obliquus, suna nuna nau'ikan tasirin induction mai yawa (Hoto na 5B). Sabanin haka, nau'ikan kamar N. purpurea da N. obtusifolia suna da martanin metabolism mara a bayyane ga magani, amma metabolome ya fi bambanta. Rarraba takamaiman nau'ikan martanin metabolism da aka haifar ya haifar da babban alaƙa mara kyau tsakanin ƙwarewa da bambancin gamma (PCC = -0.46, P = 4.9×10-8). Canje-canjen da OS ke haifarwa a matakan JAs suna da alaƙa mai kyau da ƙwarewar metabolism, kuma suna da alaƙa mara kyau da bambancin gamma na metabolism wanda kowane nau'in ya nuna (Hoto na 5B da Hoto na S9). Ya kamata a lura cewa nau'in da ake kira nau'in "amsar sigina" a cikin Hoto na 5C, kamar Nepenthes nematodes, Nepenthes nepenthes, Nepenthes acute, da Nepenthes acute, sun haifar da manyan alamu a cikin mintuna 30. Barkewar JA da JA-Ile ta OS ta kwanan nan, yayin da sauran ƙwayoyin cuta da ake kira "mara amsawa ga sigina", kamar Nepenthes mills, Nepenthes powdery da N. obtusifolia suna nuna JA-Ile Edge kawai ba tare da takamaiman OS ba (Hoto na 5C). A matakin metabolism, kamar yadda aka ambata a sama, ga Nepenthes da aka rage, abubuwan da ke amsawa ga sigina sun nuna takamaiman OS kuma sun ƙara δj sosai, yayin da suke rage Hj. Ba a gano wannan tasirin priming na musamman na OS a cikin nau'ikan da aka rarraba a matsayin nau'in sigina marasa amsawa ba (Hoto na 5, D da E). Ana raba metabolites na musamman na OS akai-akai tsakanin nau'ikan da ke amsawa ga sigina, kuma waɗannan rukunin sigina suna taruwa tare da nau'ikan da ke da raunin martanin sigina, yayin da nau'ikan da ke da raunin martanin sigina suna nuna ƙarancin dogaro da juna (Hoto na S8). Wannan sakamakon yana nuna cewa shigar da JAs na musamman na OS da sake tsara tsarin metabolome na ƙasa an haɗa su a matakin nau'in.
Na gaba, mun yi amfani da man lanolin wanda ke ɗauke da methyl JA (MeJA) don magance tsire-tsire don bincika ko waɗannan hanyoyin haɗin gwiwa suna da iyaka ta hanyar samuwar JA da JA na waje ke amfani da shi, wanda zai kasance a cikin cytoplasm na tsire-tsire. Saurin lalata ƙasa shine JA. Mun sami irin wannan yanayin na canjin hankali daga nau'ikan da ke amsawa ga sigina zuwa nau'ikan da ba sa amsawa ta hanyar ci gaba da samar da JA (Hoto na 5, D da E). A takaice, maganin MeJA ya sake tsara metabolomes na layi, N. obliquus, N. aquaticus, N. pallens, da N. mikimotoi, wanda ya haifar da ƙaruwa mai yawa a cikin δj da raguwa a cikin Hj. N. purpurea kawai ya nuna ƙaruwa a cikin δj, amma ba Hj. N. obtusifolia ba, wanda a baya aka nuna yana tara ƙananan matakan JAs, kuma yana amsawa mara kyau ga maganin MeJA dangane da sake saita metabolome. Waɗannan sakamakon sun nuna cewa samar da JA ko kuma canja wurin siginar yana da iyaka a fannin ilimin halittar jiki a cikin nau'ikan da ba sa amsawa ga siginar. Don gwada wannan hasashe, mun yi nazarin nau'ikan guda huɗu (N. pallens, N. mills, N. pink da N. microphylla) waɗanda W + W, W + OSMs da W + OSS1 Transcriptome suka haifar (39). Daidai da tsarin sake fasalin metabolome, nau'ikan sun rabu sosai a cikin sararin transcriptome, daga cikinsu N. attenuated ya nuna mafi girman RDPI da OS ya haifar, yayin da N. gracilis yana da mafi ƙanƙanta (Hoto na 6A). Duk da haka, an gano cewa bambancin transcriptome da N. oblonga ya haifar shine mafi ƙanƙanta a cikin nau'ikan guda huɗu, sabanin mafi girman bambancin metabonomic na N. oblonga da aka nuna a baya a cikin nau'ikan guda bakwai. Nazarin da suka gabata ya nuna cewa saitin kwayoyin halitta da suka shafi siginar tsaro ta farko, gami da siginar JA, yana bayyana takamaiman martanin tsaro na farko da masu haifar da herbivore ke haifarwa a cikin nau'ikan Nicotiana (39). Kwatanta hanyoyin siginar JA tsakanin waɗannan nau'ikan guda huɗu sun bayyana wani tsari mai ban sha'awa (Hoto na 6B). Yawancin kwayoyin halitta a cikin wannan hanyar, kamar AOC, OPR3, ACX da COI1, sun nuna matakan shigar da ƙwayoyin halitta a cikin waɗannan nau'ikan guda huɗu. Duk da haka, wani muhimmin kwayar halitta, JAR4, yana canza JA zuwa nau'in JA-Ile mai aiki a zahiri, kuma matakin shigar da ƙwayoyin halitta yana da ƙasa sosai, musamman a cikin N. mills, Nepenthes pieris da N. microphylla. Bugu da ƙari, ba a gano rubutun wani kwayar halitta AOS kawai a cikin N. bifidum ba. Waɗannan canje-canje a cikin bayyanar kwayoyin halitta na iya zama alhakin mummunan yanayin da ƙarancin samar da JA a cikin nau'in rashin lafiyar sigina da kuma shigar da N. gracilis ya haifar.
(A) Binciken ka'idar bayanai game da sake tsara martanin rubuce-rubuce na farko na nau'ikan taba guda huɗu masu alaƙa da aka ɗauki samfurinsu mintuna 30 bayan shigar da ganyen herbivory. Ana ƙididdige RDPI ta hanyar kwatanta ganyen da herbivory OS ya haifar da maganin rauni. Launuka suna nuna nau'ikan daban-daban, kuma alamomin suna nuna hanyoyin magani daban-daban. (B) Binciken bayyanar kwayoyin halitta a cikin hanyoyin siginar JA tsakanin nau'ikan guda huɗu. An nuna hanyar JA mai sauƙi kusa da taswirar akwatin. Launuka daban-daban suna nuna hanyoyin sarrafawa daban-daban. Alamar ta nuna cewa akwai babban bambanci tsakanin maganin W + OS da sarrafa W + W (don gwajin t na ɗalibi don bambance-bambancen biyu, *P<0.05, **P<0.01 da ***P<0.001). OPDA, 12-oxophytodienoic acid; OPC-8: 0,3-oxo-2(2′(Z)-pentenyl)-cyclopentane-1-octanoic acid.
A cikin kashi na ƙarshe, mun yi nazarin yadda sake fasalin nau'in kwari na metabolome na nau'ikan tsire-tsire daban-daban zai iya zama mai juriya ga masu cin ganyayyaki. Binciken da aka yi a baya ya jaddada nau'in Nicotiana. Juriyarsu ga Ms da tsutsotsi sun bambanta sosai (40). A nan, mun yi nazarin alaƙar da ke tsakanin wannan samfurin da ƙarfin metabolism ɗinsu. Ta amfani da nau'ikan taba guda huɗu da ke sama, da kuma gwada alaƙar da ke tsakanin bambancin da ƙwarewar metabolome da masu cin ganyayyaki ke haifarwa da juriyar tsire-tsire ga Ms da Sl, mun gano juriya, bambancin da ƙwarewa ga mai cin ganyayyaki na Sl All suna da alaƙa mai kyau, yayin da alaƙar da ke tsakanin juriya ga mata ƙwararru da ƙwarewa ba ta da ƙarfi, kuma alaƙar da ke tsakanin bambancin ba ta da mahimmanci (Hoto na S10). Dangane da juriyar S1, duka N. chinensis da N. gracilis da aka rage, waɗanda aka nuna a baya suna nuna matakan jigilar siginar JA da ƙarfin metabolome, suna da martani daban-daban ga shigar da herbivore, kuma sun kuma nuna irin wannan juriya mai girma. Jima'i.
A cikin shekaru sittin da suka gabata, ka'idar kare tsirrai ta samar da tsarin ka'ida, wanda bisa ga haka masu bincike suka yi hasashen yawan juyin halitta da ayyukan ƙwayoyin halitta na musamman na shuka. Yawancin waɗannan ka'idoji ba sa bin tsarin yau da kullun na ƙididdiga masu ƙarfi (41). Suna ba da shawarar manyan hasashe (3) a matakin bincike ɗaya. Lokacin da gwajin manyan hasashe ya ba da damar yin nazarin takamaiman ka'idoji, wannan zai sa filin yana ci gaba. A goyi bayansa, amma a ƙi wasu (42). Madadin haka, sabuwar ka'idar tana yin hasashe a matakai daban-daban na bincike kuma tana ƙara sabon matakin la'akari da bayanin (42). Duk da haka, ka'idojin guda biyu da aka gabatar a matakin aiki, MT da OD, za a iya bayyana su cikin sauƙi a matsayin muhimman hasashe na canje-canje na musamman na rayuwa da masu cin ganyayyaki ke haifarwa: Ka'idar OD ta yi imanin cewa canje-canje a cikin "sararin samaniya" na rayuwa na musamman suna da matuƙar shugabanci. Ka'idar MT ta yi imanin cewa waɗannan canje-canjen ba za su kasance ba bisa ka'ida ba kuma bazuwar su ne a cikin sararin rayuwa, kuma galibi suna da manyan ƙwayoyin halitta masu ƙimar kariya. An gwada gwaje-gwajen da suka gabata na hasashen OD da MT ta amfani da kunkuntar saitin mahaɗan "kariya" na priori. Waɗannan gwaje-gwajen da suka shafi metabolites sun hana ikon yin nazarin girman da kuma yanayin sake fasalin metabolome a lokacin herbivory, kuma ba sa barin gwaji a cikin tsarin ƙididdiga mai daidaito don buƙatar manyan hasashen da za a iya la'akari da su gaba ɗaya. Canje-canjen Quantify a cikin metabolome na shuka. A nan, mun yi amfani da fasahar zamani a cikin metabolomics bisa ga MS na lissafi kuma mun gudanar da nazarin MS na deconvolution a cikin kudin gabaɗaya na bayanin ka'idar bayanai don gwada bambanci tsakanin biyun da aka gabatar a matakin metabolomics na duniya. Babban hasashen wannan ka'idar. An yi amfani da ka'idar bayanai a fannoni da yawa, musamman a cikin mahallin binciken bambancin halittu da kwararar abinci mai gina jiki (43). Duk da haka, gwargwadon sani, wannan shine aikace-aikacen farko da aka yi amfani da shi don bayyana sararin bayanai na metabolism na shuke-shuke da kuma magance matsalolin muhalli da suka shafi canje-canje na metabolism na ɗan lokaci dangane da alamun muhalli. Musamman, ikon wannan hanyar yana cikin ikonsa na kwatanta alamu a cikin da tsakanin nau'ikan shuke-shuke don bincika yadda masu cin ganyayyaki suka canza daga nau'ikan daban-daban zuwa tsarin macroevolution tsakanin nau'ikan a matakai daban-daban na juyin halitta. Metabolism.
Babban nazarin sassa (PCA) yana canza saitin bayanai masu canzawa zuwa sararin rage girma ta yadda za a iya bayyana babban yanayin bayanan, don haka yawanci ana amfani da shi azaman dabarar bincike don tantance saitin bayanai, kamar metabolome na deconvolution. Duk da haka, rage girma zai rasa wani ɓangare na abubuwan da ke cikin bayanan, kuma PCA ba zai iya samar da bayanai masu yawa game da halaye waɗanda suka dace da ka'idar muhalli ba, kamar: yadda masu cin ganyayyaki ke sake tsara bambancin a fannoni na musamman (misali, wadata, rarrabawa) Da yalwa) metabolites? Waɗanne metabolites ne masu hasashen yanayin da aka haifar da wani mai cin ganyayyaki? Daga mahangar takamaiman, bambancin da rashin iyawa, abubuwan da ke cikin bayanin bayanin metabolite na takamaiman ganye ya lalace, kuma an gano cewa cin masu cin ganyayyaki na iya kunna takamaiman metabolism. Ba zato ba tsammani, mun lura cewa, kamar yadda aka bayyana a cikin alamun ka'idar bayanai da aka aiwatar, yanayin metabolism da ya haifar yana da babban haɗuwa bayan hare-haren masu cin ganyayyaki biyu (mai cin ganyayyaki na dare Sl) da ƙwararren Solanaceae Ms. Kodayake halayen ciyar da su da maida hankali sun bambanta sosai. Mai ƙaddamar da sinadarin mai mai-amino acid conjugate (FAC) a cikin OS (31). Ta hanyar amfani da herbivore OS don magance raunukan hudawa na yau da kullun, maganin herbivore da aka yi kwaikwayon shi ma ya nuna irin wannan yanayin. Wannan tsari na daidaitawa don kwaikwayon martanin tsire-tsire ga hare-haren herbivore yana kawar da abubuwan da ke haifar da sauye-sauye a cikin halayen cin abincin herbivore, wanda ke haifar da lalacewa daban-daban a lokuta daban-daban (34). FAC, wanda aka san shine babban dalilin OSM, yana rage JAS da sauran martanin hormone na shuka a cikin OSS1, yayin da OSS1 ke raguwa sau ɗaruruwa (31). Duk da haka, OSS1 ya haifar da irin wannan matakan tarin JA idan aka kwatanta da OSM. An riga an nuna cewa martanin JA a cikin Nepenthes da aka rage yana da matukar tasiri ga OSM, inda FAC zai iya ci gaba da aikinsa ko da an narkar da shi da ruwa 1:1000 (44). Saboda haka, idan aka kwatanta da OSM, kodayake FAC a cikin OSS1 yana da ƙasa sosai, ya isa ya haifar da isasshen barkewar JA. Nazarin da aka yi a baya ya nuna cewa ana iya amfani da sunadaran porin-like (45) da oligosaccharides (46) a matsayin alamun kwayoyin halitta don haifar da martanin kare tsirrai a cikin OSS1. Duk da haka, har yanzu ba a san ko waɗannan abubuwan da ke haifar da cutar a cikin OSS1 suna da alhakin tarin JA da aka lura a cikin binciken na yanzu ba.
Duk da cewa akwai ƙananan bincike da ke bayyana bambancin yatsan metabolism da aka samu sakamakon amfani da ganyen ciyawa daban-daban ko kuma JA ko SA na waje (salicylic acid) (47), babu wanda ya dame shi da tasirin takamaiman nau'in ganyen herbivore a cikin hanyar sadarwa ta ciyawar shuka da tasirinsa akan takamaiman. Ana nazarin tasirin metabolism gabaɗaya cikin tsari. Bayanan sirri. Wannan binciken ya ƙara tabbatar da cewa haɗin hanyar sadarwa ta hormone na ciki da wasu hormones na shuka banda JA yana siffanta takamaiman sake tsara metabolism da ganyen herbivore ke haifarwa. Musamman, mun gano cewa ET da OSM ke haifarwa ya fi wanda OSS1 ke haifarwa girma. Wannan yanayin ya yi daidai da ƙarin abubuwan FAC a cikin OSM, wanda shine yanayi mai mahimmanci kuma isasshe don haifar da fashewar ET (48). A cikin mahallin hulɗar tsakanin tsire-tsire da ganyen herbivore, aikin siginar ET akan yanayin metabolite na musamman na shuka har yanzu yana faruwa lokaci-lokaci kuma yana kai hari ga rukuni ɗaya kawai. Bugu da ƙari, yawancin bincike sun yi amfani da amfani da ET ko abubuwan da suka riga suka fara aiki ko kuma wasu masu hana su daban-daban don nazarin ƙa'idar ET, daga cikinsu waɗannan aikace-aikacen sinadarai na waje za su haifar da illoli da yawa marasa takamaiman takamaiman sakamako. A fahimtarmu, wannan binciken yana wakiltar babban bincike na farko na tsarin aiki na ET a cikin amfani da ET don samar da kuma fahimtar tsire-tsire masu canza halitta don daidaita yanayin metabolism na shuka. Induction na musamman na ET na herbivore zai iya daidaita martanin metabolome. Mafi mahimmanci shine sarrafa kwayoyin halittar ET na transgenic (ACO) da tsinkaye (ETR1) waɗanda suka bayyana tarin phenolamides na musamman na herbivore. An riga an nuna cewa ET na iya daidaita tarin nicotine da JA ta haifar ta hanyar daidaita putrescine N-methyltransferase (49). Duk da haka, daga mahangar injiniya, ba a bayyana yadda ET ke daidaita shigar da phenamide ba. Baya ga aikin transduction na siginar ET, ana iya karkatar da kwararar metabolism zuwa S-adenosyl-1-methionine don daidaita saka hannun jari a cikin polyaminophenol amides. S-adenosyl-1-methionine shine ET kuma matsakaici ne na hanyar biosynthetic na polyamine. Tsarin da siginar ET ke sarrafa matakin phenolamide yana buƙatar ƙarin bincike.
Na dogon lokaci, saboda yawan metabolites na musamman na tsarin da ba a san shi ba, tsananin kulawa ga takamaiman nau'ikan metabolism bai iya tantance canje-canje na ɗan lokaci na bambancin metabolism bayan hulɗar halittu ba. A halin yanzu, bisa ga nazarin ka'idar bayanai, babban sakamakon samun MS/MS bakan bisa ga metabolites marasa son kai shine cewa masu cin ganyayyaki waɗanda ke cin abinci ko kwaikwayon masu cin ganyayyaki suna ci gaba da rage bambancin metabolism na metabolome na ganye yayin da suke ƙara matakin ƙwarewa. Wannan ƙaruwa na ɗan lokaci a cikin takamaiman metabolome da masu cin ganyayyaki ke haifarwa yana da alaƙa da ƙaruwar haɗin gwiwa a cikin takamaiman transcriptome. Siffar da ta fi ba da gudummawa ga wannan babban ƙwarewar metabolome (yana da ƙimar Si mafi girma) ita ce metabolite na musamman tare da aikin herbivorous da aka riga aka siffanta. Wannan samfurin ya yi daidai da hasashen ka'idar OD, amma hasashen MT da ke da alaƙa da bazuwar sake tsara metabolome ba daidai ba ne. Duk da haka, wannan bayanan kuma ya yi daidai da hasashen gaurayen samfurin (mafi kyawun MT; Hoto na 1B), saboda sauran metabolites marasa siffa tare da ayyukan tsaro da ba a san su ba har yanzu suna iya bin rarraba Si bazuwar.
Wani abin lura da wannan binciken ya gano shi ne, daga matakin ƙananan halittu (yawan tsire-tsire da taba) zuwa babban sikelin juyin halitta (nau'in taba masu alaƙa da juna), matakai daban-daban na tsarin juyin halitta suna cikin "mafi kyawun kariya". Akwai manyan bambance-bambance a cikin iyawar masu cin ganyayyaki. Moore et al. (20) da Kessler da Kalske (1) sun ba da shawarar canza matakan aiki guda uku na bambancin halittu waɗanda Whittaker (50) ya bambanta zuwa canje-canje na lokaci-lokaci da suka haifar da bambancin sinadarai; waɗannan marubutan ba su taƙaita ba. Tsarin tattara bayanai na metabolome mai girma kuma ba su bayyana yadda ake ƙididdige bambancin metabolism daga waɗannan bayanan ba. A cikin wannan binciken, ƙananan gyare-gyare ga rarrabuwar aikin Whittaker za su yi la'akari da bambancin α-metabolic a matsayin bambancin MS/MS a cikin wani shuka da aka bayar, da kuma bambancin β-metabolic a matsayin babban metabolism na intraspecific na ƙungiyar al'umma Sarari, da bambancin γ-metabolic zai zama faɗaɗa nazarin nau'ikan halittu iri ɗaya.
Siginar JA tana da mahimmanci ga nau'ikan martanin metabolism na herbivores iri-iri. Duk da haka, akwai rashin gwaji mai tsauri na yawan gudummawar da ke tattare da tsarin JA na halitta ga bambancin metabolome, kuma ko siginar JA wuri ne na gabaɗaya don bambancin metabolism wanda damuwa ke haifarwa akan babban sikelin macroevolutionary har yanzu ba a iya fahimta ba. Mun lura cewa yanayin herbivores na Nepenthes herbivores yana haifar da ƙwarewar metabolome kuma bambancin ƙwarewar metabolome a cikin yawan nau'ikan Nicotiana da tsakanin nau'ikan Nicotiana masu alaƙa yana da alaƙa mai kyau tare da siginar JA. Bugu da ƙari, lokacin da siginar JA ta lalace, takamaiman metabolism da herbivore guda ɗaya ya haifar za a soke shi (Hoto na 3, C da E). Tunda canje-canjen bakan metabolism na yawan Nepenthes da aka rage a zahiri galibi adadi ne, canje-canje a cikin bambancin β na metabolism da takamaiman aiki a cikin wannan bincike na iya zama galibi saboda ƙarfin kuzari na nau'ikan mahadi masu wadataccen metabolite. Waɗannan azuzuwan mahadi sun mamaye wani ɓangare na bayanin metabolome kuma suna haifar da kyakkyawar alaƙa da siginar JA.
Saboda hanyoyin sinadarai na nau'ikan taba da ke da alaƙa da ita sun bambanta sosai, ana gano metabolites musamman a ɓangaren inganci, don haka yana da ƙarin nazari. Tsarin ka'idar bayanai na bayanin metabolism da aka kama ya nuna cewa haifar da herbivorous yana ƙara ta'azzara ciniki tsakanin bambancin gamma na metabolism da ƙwarewa. Siginar JA tana taka muhimmiyar rawa a cikin wannan ciniki. Ƙarawar ƙwarewar metabolome ya yi daidai da babban hasashen OD kuma yana da alaƙa mai kyau da siginar JA, yayin da siginar JA tana da alaƙa mara kyau da bambancin gamma na metabolism. Waɗannan samfuran suna nuna cewa ƙarfin OD na tsire-tsire galibi ana ƙaddara shi ta hanyar ƙarfin JA, ko a kan sikelin microevolutionary ko a kan babban sikelin juyin halitta. Gwaje-gwajen aikace-aikacen JA na waje waɗanda ke kawar da lahani na biosynthesis na JA sun ƙara nuna cewa nau'ikan taba masu alaƙa za a iya bambanta su zuwa nau'ikan masu amsawa da sigina da marasa amsawa, kamar yanayin JA da ƙarfin metabolome da herbivores ke haifarwa. Nau'ikan sigina marasa amsawa ba za su iya amsawa ba saboda rashin iyawarsu na samar da JA na ciki kuma saboda haka suna ƙarƙashin iyakokin ilimin halittar jiki. Wannan na iya faruwa ne sakamakon maye gurbi a cikin wasu manyan kwayoyin halitta a cikin hanyar siginar JA (AOS da JAR4 a cikin N. crescens) na. Wannan sakamakon ya nuna cewa waɗannan nau'ikan macroevolutionary na iya zama galibi sakamakon canje-canje a cikin fahimtar hormones na ciki da amsawar su.
Baya ga hulɗar da ke tsakanin tsirrai da masu cin ganyayyaki, binciken bambancin rayuwa yana da alaƙa da duk wani muhimmin ci gaba na ka'ida a cikin binciken daidaitawar halittu ga muhalli da kuma juyin halittar halaye masu rikitarwa na siffofi. Tare da ƙaruwar adadin bayanai da kayan aikin MS na zamani suka samu, gwajin hasashen bambancin rayuwa yanzu zai iya wuce bambance-bambancen metabolite na mutum/rukuni da kuma yin bincike na duniya don bayyana alamu marasa tsammani. A cikin tsarin babban bincike, wani muhimmin kwatanci shine ra'ayin ƙirƙirar taswira masu ma'ana waɗanda za a iya amfani da su don bincika bayanai. Saboda haka, muhimmin sakamako na haɗuwar metabolomics na MS/MS da ka'idar bayanai mara son kai shine yana samar da ma'auni mai sauƙi wanda za a iya amfani da shi don gina taswira don bincika bambancin rayuwa akan ma'aunin taxonomic daban-daban. Wannan shine babban buƙatar wannan hanyar. Nazarin juyin halittar micro/macro da ilimin halittu na al'umma.
A matakin juyin halitta na macro-evolutionary, tushen ka'idar juyin halitta tsakanin tsirrai da kwari na Ehrlich da Raven (51) shine a annabta cewa bambancin bambancin rayuwa tsakanin nau'ikan halittu shine dalilin bambancin jinsin tsirrai. Duk da haka, a cikin shekaru hamsin tun bayan buga wannan aikin na musamman, ba a gwada wannan hasashen ba sosai (52). Wannan ya faru ne saboda halayen phylogenetic na halaye masu kama da juna na rayuwa a cikin layin tsirrai masu nisa. Ana iya amfani da ƙarancin don daidaita hanyoyin nazarin manufa. Aikin MS/MS na yanzu wanda aka sarrafa ta ka'idar bayanai yana ƙididdige kamanceceniya na tsarin MS/MS na metabolites da ba a sani ba (ba tare da zaɓin metabolite na baya ba) kuma yana canza waɗannan MS/MS zuwa saitin MS/MS, don haka a cikin metabolism na ƙwararru Ana kwatanta waɗannan samfuran macro-evolutionary a cikin sikelin rarrabuwa. Ma'aunin ƙididdiga masu sauƙi. Tsarin yana kama da nazarin phylogenetic, wanda zai iya amfani da daidaitawar jeri don ƙididdige yawan bambancin jinsi ko juyin halittar hali ba tare da annabta ta gaba ba.
A matakin sinadarai masu rai, hasashen tantancewa na Firn da Jones (53) ya nuna cewa ana kiyaye bambancin metabolism a matakai daban-daban don samar da kayan aiki don aiwatar da ayyukan halittu na metabolites marasa alaƙa ko waɗanda aka maye gurbinsu a baya. Hanyoyin ka'idar bayanai suna ba da tsarin da za a iya ƙididdige waɗannan sauye-sauyen juyin halitta na metabolites waɗanda ke faruwa a lokacin ƙwarewar metabolites a matsayin wani ɓangare na tsarin tantance juyin halitta da aka gabatar: daidaitawa ta hanyar halitta daga ƙarancin takamaiman zuwa babban takamaiman metabolites da aka hana na wani yanayi da aka bayar.
Gabaɗaya, a farkon zamanin ilmin halittar kwayoyin halitta, an ƙirƙiri muhimman ka'idojin kare tsirrai, kuma ana ɗaukar hanyoyin da aka dogara da hasashen ragewa a matsayin hanya ɗaya tilo ta ci gaban kimiyya. Wannan ya faru ne saboda iyakokin fasaha na auna dukkan metabolome. Duk da cewa hanyoyin da aka dogara da hasashen suna da amfani musamman wajen zaɓar wasu hanyoyin dalili, ikonsu na haɓaka fahimtarmu game da hanyoyin sadarwa na sinadarai ya fi iyaka fiye da hanyoyin lissafi da ake da su a yanzu a kimiyyar zamani mai zurfin bayanai. Saboda haka, ka'idojin da ba za a iya annabta ba sun fi ƙarfin iyakokin bayanai da ake da su, don haka ba za a iya kawar da dabarar hasashe/zagayen gwaji na ci gaba a fagen bincike ba (4). Mun hango cewa tsarin aiki na lissafi na metabolomics da aka gabatar a nan na iya sake farfaɗo da sha'awa game da batutuwan da suka gabata (ta yaya) da na ƙarshe (dalilin) ​​na bambancin metabolism, da kuma ba da gudummawa ga sabon zamani na kimiyyar bayanai da aka jagoranta a ka'ida. Zamanin ya sake duba mahimman ka'idojin da suka yi wahayi zuwa ga tsararraki na baya.
Ana yin ciyar da tsuntsayen da ke cin ciyawa kai tsaye ta hanyar ɗaga tsutsa ta biyu a kan ganyen shuka mai launin shuɗi na shukar fure ɗaya mai fure, tare da kwafi iri 10 na shuka a kowace shuka. An daure tsutsar kwari da manne, kuma an tattara sauran kyallen ganyen bayan sa'o'i 24 da 72 bayan kamuwa da cuta kuma an daskare su da sauri, sannan aka cire metabolites ɗin.
Yi kwaikwayon maganin ganye ta hanyar da aka haɗa sosai. Hanyar ita ce a yi amfani da ƙafafun zane don huda layuka uku na ƙaya a kowane gefen tsakiyar ganyen shuka guda uku da aka faɗaɗa gaba ɗaya a lokacin girman furen yadin, sannan a shafa nan da nan Ms. An narkar da shi da ruwa 1:5 Ko kuma a yi amfani da yatsun hannu don saka S1 OS a cikin raunin huda. Girbe kuma a sarrafa ganyen kamar yadda aka bayyana a sama. Yi amfani da hanyar da aka bayyana a baya don cire manyan metabolites da hormones na shuka (54).
Don amfani da ganyen JA na waje, ana yi wa ganyen petiole guda uku na kowanne daga cikin tsire-tsire shida masu fure na kowanne nau'in magani da 20μl na manna lanolin wanda ke ɗauke da 150μg MeJA (Lan + MeJA), da 20μl na lanolin da maganin raunuka (Lan + W), ko kuma a yi amfani da 20μl tsantsar lanolin a matsayin magani. An girbe ganyen awanni 72 bayan magani, an daskare su a cikin ruwa mai nitrogen, sannan a adana su a -80°C har sai an yi amfani da su.
An gano layukan JA da ET guda huɗu na transgenic, wato irAOC (36), irCOI1 (55), irACO da sETR1 (48), a cikin ƙungiyar bincikenmu. irAOC ya nuna raguwa sosai a matakan JA da JA-Ile, yayin da irCOI1 bai nuna damuwa ga JAs ba. Idan aka kwatanta da EV, tarin JA-Ile ya ƙaru. Hakazalika, irACO zai rage samar da ET, kuma idan aka kwatanta da EV, sETR1, wanda ba shi da hankali ga ET, zai ƙara samar da ET.
Ana amfani da na'urar auna haske ta laser (Sensor Sense ETD-300 real-time ET firikwensin) don auna ET ba tare da yin ɓarna ba. Nan da nan bayan an yi magani, an yanke rabin ganyen kuma an mayar da su cikin kwalbar gilashi mai rufi mai 4-ml, kuma an bar sararin kan ya taru cikin awanni 5. A lokacin aunawa, an wanke kowace kwalba da ruwa mai lita 2/awa na iska mai tsabta na tsawon mintuna 8, wanda a baya ya wuce ta cikin wani abu mai kara kuzari da Sensor Sense ya samar don cire CO2 da ruwa.
An fara buga bayanan microarray a cikin (35) kuma an adana su a cikin Cibiyar Bayar da Bayanin Halitta ta Ƙasa (NCBI) Cikakken Bayanan Halittar Bayyanar Halitta (lambar shiga GSE30287). An cire bayanan da suka dace da ganyen da maganin W + OSMs ya haifar da kuma ikon sarrafawa mara lalacewa don wannan binciken. Ƙarfin da ba a iya amfani da shi ba shine log2. Kafin nazarin ƙididdiga, an canza tushen kuma an daidaita shi zuwa kashi 75 cikin ɗari ta amfani da fakitin software na R.
An samo asalin bayanan jerin RNA (RNA-seq) na nau'in Nicotiana daga Tarihin Karatu na NCBI (SRA), lambar aikin ita ce PRJNA301787, wanda Zhou et al. (39) suka ruwaito kuma suka ci gaba kamar yadda aka bayyana a cikin (56). An zaɓi bayanan da aka sarrafa ta hanyar W + W, W + OSM da W + OSS1 waɗanda suka dace da nau'in Nicotiana don bincike a cikin wannan binciken, kuma an sarrafa su ta hanyar da ke ƙasa: Da farko, an canza karatun RNA-seq na asali zuwa tsarin FASTQ. HISAT2 yana canza FASTQ zuwa SAM, kuma SAMtools yana canza fayilolin SAM zuwa fayilolin BAM da aka tsara. Ana amfani da StringTie don ƙididdige bayyanar kwayar halitta, kuma hanyar bayyana ta ita ce akwai gutsuttsura ga gutsuttsura dubu na tushe ga gutsuttsura miliyan da aka jera.
Ginshiƙin chromatographic na Acclaim (150 mm x 2.1 mm; girman barbashi 2.2μm) da aka yi amfani da shi a cikin binciken da ginshiƙin tsaro na 4 mm x 4 mm ya ƙunshi abu ɗaya. Ana amfani da wannan ginshiƙin binary a cikin tsarin Dionex UltiMate 3000 Ultra High Performance Liquid Chromatography (UHPLC): mintuna 0 zuwa 0.5, isocratic 90% A [ruwa mai narkewa, 0.1% (v/v) acetonitrile da 0.05% formic acid], 10% B (Acetonitrile da 0.05% formic acid); mintuna 0.5 zuwa 23.5, matakin gradient shine 10% A da 90% B, bi da bi; mintuna 23.5 zuwa 25, isocratic 10% A da 90% B. Yawan kwarara shine 400μl/min. Ga duk nazarin MS, a yi allurar ginshiƙin da ke sama a cikin na'urar auna lokaci-lokaci da kuma lokacin tashi (qTOF) wacce aka sanya matattarar feshi ta lantarki da ke aiki a yanayin ionization mai kyau (ƙarfin wutar lantarki na capillary, 4500 V; mashigar capillary 130 V; zafin bushewa 200°C; busar da iska lita 10/min).
Yi nazarin guntu na MS / MS (wanda daga baya ake kira MS / MS) wanda ba shi da mahimmanci ko kuma ba za a iya bambance shi da bayanai ba don samun bayanai game da tsarin tsarin gabaɗayan bayanin metabolism da za a iya gano shi. Manufar hanyar MS/MS ba tare da la'akari da juna ta dogara ne akan gaskiyar cewa quadrupole yana da babban taga na ware taro [saboda haka, yi la'akari da duk siginar taro-zuwa-caji (m/z) a matsayin gutsuttsura]. Saboda wannan dalili, saboda kayan aikin Impact II bai iya ƙirƙirar karkatar CE ba, an yi bincike mai zaman kansa da yawa ta amfani da ƙimar kuzarin karo na karo da aka haifar da karo (CE). A takaice, da farko bincika samfurin ta hanyar UHPLC-electrospray ionization/qTOF-MS ta amfani da yanayin spectrometry na taro ɗaya (ƙananan yanayin rarrabuwa da aka samar ta hanyar rarrabuwar tushen), duba daga m/z 50 zuwa 1500 a mita maimaituwa na 5 Hz. Yi amfani da nitrogen azaman iskar gas don nazarin MS/MS, kuma yi ma'auni masu zaman kansu a waɗannan ƙarfin lantarki guda huɗu daban-daban da karo ya haifar: 20, 30, 40, da 50 eV. A duk tsawon tsarin aunawa, quadrupole yana da mafi girman taga warewar taro, daga m/z 50 zuwa 1500. Lokacin da aka saita gwajin m/z na gaba da faɗin warewar jiki zuwa 200, ana kunna kewayon taro ta atomatik ta hanyar software na aiki na kayan aiki da 0 Da. Duba guntu-guntu na taro kamar yadda yake a cikin yanayin taro ɗaya. Yi amfani da sodium formate (isopropanol 50 ml, 200 μl formic acid da 1 ml 1M NaOH maganin ruwa) don daidaita taro. Ta amfani da algorithm na Bruker mai daidaito, ana daidaita fayil ɗin bayanai bayan gudanar da matsakaicin bakan a cikin wani lokaci da aka ƙayyade. Yi amfani da aikin fitarwa na software na Nazarin Bayanai v4.0 (Brook Dalton, Bremen, Jamus) don canza fayilolin bayanai na asali zuwa tsarin NetCDF. An adana saitin bayanai na MS/MS a cikin bayanan metabolomics na buɗe MetaboLights (www.ebi.ac.uk) tare da lambar shiga. MTBLS1471.
Ana iya cimma haɗakar MS/MS ta hanyar nazarin alaƙa tsakanin siginar ingancin MS1 ​​da MS/MS don ƙarancin kuzarin karo da babban ƙarfi da sabbin ƙa'idodi da aka aiwatar. Ana amfani da rubutun R don fahimtar nazarin alaƙar rarrabawa na precursor zuwa samfurin, kuma ana amfani da rubutun C# (https://github.com/MPI-DL/indiscriminant-MS-MS-assembly-pipeline) don aiwatar da ƙa'idodin.
Domin rage kurakuran ƙarya masu kyau da hayaniyar bango ke haifarwa da kuma haɗin ƙarya da ke haifarwa ta hanyar gano wasu fasalulluka na m/z a cikin samfura kaɗan kawai, muna amfani da aikin "cikakken kololuwa" na fakitin R XCMS (don gyaran hayaniyar bango). Ya kamata a yi amfani da shi don maye gurbin ƙarfin "NA" (kololuwa da ba a gano ba). Lokacin da aka yi amfani da aikin kololuwa na cikawa, har yanzu akwai ƙimar ƙarfi "0" da yawa a cikin saitin bayanai waɗanda za su shafi lissafin haɗin gwiwa. Sannan, muna kwatanta sakamakon sarrafa bayanai da aka samu lokacin da aka yi amfani da aikin kololuwa da kuma lokacin da ba a yi amfani da aikin kololuwa da aka cika ba, kuma muna ƙididdige ƙimar hayaniyar bango bisa ga matsakaicin ƙimar da aka gyara, sannan mu maye gurbin waɗannan ƙimar ƙarfi 0 tare da ƙimar bango da aka ƙididdige. Mun kuma yi la'akari da fasalulluka waɗanda ƙarfinsu ya wuce ƙimar bango sau uku kuma muka ɗauke su a matsayin "kololuwa na gaske." Don lissafin PCC, ana la'akari da siginar m/z na samfurin precursor (MS1) da saitin bayanan guntu tare da aƙalla kololuwa na gaskiya takwas kawai.
Idan ƙarfin fasalin ingancin farko a cikin samfurin gaba ɗaya yana da alaƙa sosai da raguwar ƙarfin fasalin inganci iri ɗaya wanda ke fuskantar ƙarancin kuzarin karo ko babba, kuma ba a yiwa fasalin lakabi da kololuwar isotope ta CAMERA ba, za a iya ƙara bayyana shi. Sannan, ta hanyar ƙididdige duk nau'ikan samfuran farko da za a iya haɗawa cikin daƙiƙa 3 (taga na lokacin riƙewa da aka kiyasta don riƙewa mafi girma), ana yin nazarin haɗin gwiwa. Sai lokacin da ƙimar m/z ta yi ƙasa da ƙimar farko kuma rarrabuwar MS/MS ta faru a cikin wurin samfurin iri ɗaya a cikin saitin bayanai kamar precursor wanda aka samo shi, za a ɗauke shi a matsayin guntu.
Bisa ga waɗannan ƙa'idodi guda biyu masu sauƙi, mun ware ƙa'idodin da aka ƙayyade waɗanda ke da ƙimar m/z mafi girma fiye da m/z na farkon da aka gano, kuma bisa ga matsayin samfurin inda farkon ya bayyana da kuma ɓangaren da aka ƙayyade. Hakanan yana yiwuwa a zaɓi fasalulluka masu inganci waɗanda aka samar ta hanyar guntu-guntu da yawa a cikin yanayin MS1 ​​a matsayin farkon masu takara, don haka suna samar da mahaɗan MS/MS masu yawa. Domin rage wannan yawan bayanai, idan kamannin NDP na bakan ya wuce 0.6, kuma sun kasance cikin "pcgroup" na chromatogram da CAMERA ta yi bayani, za mu haɗa su. A ƙarshe, mun haɗa dukkan sakamakon CE guda huɗu da ke da alaƙa da farkon da gutsuttsura zuwa bakan haɗin da aka yanke ta ƙarshe ta hanyar zaɓar mafi girman kololuwar ƙarfi tsakanin duk kololuwar masu takara tare da ƙimar m/z iri ɗaya a kuzarin karo daban-daban. Matakan sarrafawa na gaba sun dogara ne akan ra'ayin bakan haɗin kuma suna la'akari da yanayin CE daban-daban da ake buƙata don haɓaka yuwuwar rarrabuwa, saboda wasu gutsuttsura ana iya gano su ne kawai a ƙarƙashin takamaiman kuzarin karo.
An yi amfani da RDPI (30) don ƙididdige ƙarfin bayanin martabar metabolism. Bambancin bakan metabolism (Hj index) ya samo asali ne daga yawan abubuwan da suka fara MS/MS ta amfani da Shannon entropy na rarraba mitar MS/MS ta amfani da lissafin da ke ƙasa wanda Martínez et al. (8) suka bayyana. Hj = ∑i = 1mPijlog2(Pij) inda Pij ya yi daidai da mitar i-th MS/MS a cikin samfurin j-th (j = 1, 2,…, m) (i = 1, 2, …, m) t).
An bayyana takamaiman yanayin rayuwa (Si index) a matsayin asalin bayyanar MS/MS dangane da mita tsakanin samfuran da ake la'akari da su. Ana ƙididdige takamaiman MS/MS a matsayin Si = 1t (∑j = 1tPijPilog2PijPi)
Yi amfani da dabarar da ke ƙasa don auna ma'aunin δj na musamman na metabolome na kowane samfurin j, da matsakaicin takamaiman MS/MS δj = ∑i = 1mPijSi
An daidaita siginar MS/MS a nau'i biyu, kuma ana ƙididdige kamanceceniya bisa ga maki biyu. Da farko, ta amfani da daidaitaccen NDP (wanda kuma aka sani da hanyar haɗin cosine), yi amfani da lissafin da ke ƙasa don auna kamanceceniya tsakanin siginar NDP = (∑iS1 & S2WS1, iWS2, i) 2∑iWS1, i2∑iWS2, i2 inda S1 da S2 Daidai da haka, don siginar 1 da siginar 2, da kuma WS1, i da WS2, i yana wakiltar nauyi bisa ga ƙarfin kololuwa cewa bambancin kololuwar i-th tsakanin siginar biyu bai kai 0.01 Da ba. An ƙididdige nauyin kamar haka: W = [ƙarfin kololuwa] m [inganci] n, m = 0.5, n = 2, kamar yadda MassBank ya ba da shawara.
An aiwatar da wata hanyar maki ta biyu, wadda ta ƙunshi nazarin NL da aka raba tsakanin MS/MS. Don wannan dalili, mun yi amfani da jerin NL 52 da aka saba gani a lokacin tsarin rarrabuwar MS a lokaci guda, da kuma takamaiman NL (fayil ɗin bayanai S1) wanda aka riga aka yi bayani a kai don bakan MS/MS na metabolites na biyu na nau'in Nepenthes da aka raunana (9, 26). Ƙirƙiri vector binary na 1 da 0 ga kowane MS/MS, wanda ya dace da halin yanzu da rashin wanzuwar wasu NL bi da bi. Dangane da kamanceceniya ta nisan Euclidean, ana ƙididdige makin kamanceceniya na NL ga kowane nau'in vectors binary NL.
Don yin rukuni biyu, mun yi amfani da fakitin R DiffCoEx, wanda ya dogara ne akan faɗaɗa Nazari na Haɗakar Halitta Mai Nauyi (WGCNA). Ta amfani da matrices na maki na NDP da NL na MS/MS spectra, mun yi amfani da DiffCoEx don ƙididdige matrix na haɗin kai. Ana yin rukuni biyu ta hanyar saita sigar "cutreeDynamic" zuwa hanya = "hybrid", cutHeight = 0.9999, deepSplit = T, da minClusterSize = 10. An sauke lambar tushe ta R na DiffCoEx daga ƙarin fayil 1 ta Tesson et al. (57); Ana iya samun fakitin software na R WGCNA da ake buƙata a cikin https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA.
Domin yin nazarin hanyar sadarwa ta kwayoyin halitta ta MS/MS, mun ƙididdige haɗin haɗin kai bisa ga nau'ikan kamanceceniya na NDP da NL, kuma mun yi amfani da software na Cytoscape don ganin yanayin hanyar sadarwa ta amfani da tsarin halitta a cikin aikace-aikacen faɗaɗa tsarin CyFilescape yFiles.
Yi amfani da sigar R 3.0.1 don yin nazarin ƙididdiga akan bayanan. An tantance mahimmancin ƙididdiga ta amfani da nazarin bambance-bambancen hanyoyi biyu (ANOVA), sannan aka biyo bayan gwajin Tukey mai mahimmanci (HSD) bayan hoc. Domin a yi nazarin bambanci tsakanin maganin ganye da kuma sarrafawa, an yi nazarin rarrabawar ƙungiyoyin samfurori guda biyu masu bambancin iri ɗaya ta amfani da gwajin t na ɗalibi.
Don ƙarin kayan aiki don wannan labarin, da fatan za a duba http://advances.sciencemag.org/cgi/content/full/6/24/eaaz0381/DC1
Wannan wani labari ne da aka rarraba a ƙarƙashin sharuɗɗan Lasisin Creative Commons Attribution-Non-Commercial, wanda ke ba da damar amfani, rarrabawa da sake bugawa a kowace hanya, matuƙar amfani na ƙarshe ba don ribar kasuwanci ba ne kuma hujjar ita ce ainihin aikin daidai ne. Nassoshi.
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Ana amfani da wannan tambayar don gwada ko kai baƙo ne kuma hana aika saƙonnin banza ta atomatik.
Ka'idar bayanai tana samar da kuɗi na duniya baki ɗaya don kwatanta metabolomes na musamman da kuma hasashen ka'idojin kariya daga gwaji.
Ka'idar bayanai tana samar da kuɗi na duniya baki ɗaya don kwatanta metabolomes na musamman da kuma hasashen ka'idojin kariya daga gwaji.
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