本文主要研究内容
作者林丹(2019)在《独脚金内脂生物合成和信号传导在调控水稻根际微生物和应对稻瘟病菌机理研究》一文中研究指出:水稻(Oryza sativa L.)作为主要的粮食作物之一,满足了世界人口约一半的粮食需求。预计到2050年,全球人口将增长到约90亿。因此,为了满足这一增长需求,提高水稻产量至关重要。然而,由于稻瘟病菌(Magnaporthe oryzae)引起的稻瘟病导致全球每年水稻产量损失严重(约10-30%),全球粮食安全问题依然受到威胁。通常使用不同的杀菌剂来控制稻瘟病,例如三环唑、丙烯唑、嘧菌酯、丙环唑和异丙醇。这些杀菌剂虽然治病率高、见效快,但是化学杀菌剂的不适当或过度使用,可能对人类健康造成潜在危机,威胁生态环境。利用具有“种族特异性”抗病的作物品种是另一种可靠的疾病治理方法。但是,由于不断克服宿主防御的稻瘟病新型病原体的出现,抗病性并不持久,只能持续两三年左右。因此,迫切需要能够提高水稻对稻瘟病菌免疫力的方法以获得对稻瘟病更持久的抗性。为了抵御各种各样的生物和非生物胁迫,植物不仅激活基础防御反应,包括防御激素的产生,还依赖于根际微生物的生存和适应能力。根际微生物通过参与宿主养分获取和环境压力恢复,使其成为以生态可持续方式提高农业收益的理想方法。有趣的是,特定的植物激素和相关的信号不仅作为植物生长和防御调节因子,而且在选择特定的根际微生物成分方面发挥着积极的作用。因此,探索植物激素在防御中的作用,确定根际微生物及其相关机制,将为生态可持续方式提高作物产量提供新的途径。独脚金内酯(SLs)是一类植物激素,在植物发育和生理过程的调节中起关键作用。此外,新的证据表明,SLs还可以促进植物的特定生物和非生物抗性。SLs还充当根际信号因子;从而积极调节寄主植物与丛枝菌根真菌之间的共生关系。此外,最近还报道了SLs对根际微生物群落组成具有调节作用。这些发现表明了植物与微生物相互作用过程中SLs的潜力。然而,据我们所知,水稻在稻瘟病菌的影响下与根际微生物群落之间相互作用过程中,SLs生物合成和信号的可能调节作用和机制尚不清楚。因此,在这项研究中,我们研究了水稻在稻瘟病菌的影响下与根际微生物群落之间相互作用过程中SLs生物合成和信号的调节作用。首先,我们通过SLs生物合成或信号传导中受损的水稻突变体及其相应的野生型(WT;Oryza sativa L.cv.Shiokari)的比较分析,在稻瘟病菌感染水稻植物的过程中,探讨了SLs生物合成和信号在防御稻瘟病中的作用。结果表明,如果SLs生物合成突变体(dwarf17,d17)或信号传导突变体(d14)会导致水稻对稻瘟病菌的易感性增加。这清楚地表明SLs生物合成和信号传导能够提高对稻瘟病的抗性。为了提高水稻对稻瘟病菌的抗性,探讨了SLs调节的下游基因/途径,对d14突变体和野生型水稻植株(有或无M.oryzae感染)进行了全基因组比较叶转录组学(基于Illumina高通量测序的RNA测序)分析。然后,使用MapMan分析转录组数据并获得生物胁迫途径。基于MapMan的转录组数据集的生物胁迫显示,在水稻稻瘟病菌感染期间,d14突变体叶片中相当数量的防御相关基因(包括细胞壁、乙烯、过氧化氢合成相关基因)相对于野生植物明显受到抑制。此外,KEGG富集分析表明,与野生型水稻相比,d14突变体中的代谢途径主要涉及碳水化合物/糖代谢,包括淀粉和蔗糖代谢、戊糖磷酸途径、乙醛酸和二羧酸代谢以及光合作用在稻瘟病感染期间被显著抑制。通过分析与乙烯生物合成,H2O2生物合成和碳水化合物合成途径相关的8个基因的表达模式以及使用荧光定量PCR(qRT-PCR)的验证7个随机选择的基因来验证转录组数据结果。总的来说,qRT-PCR获得的结果与RNA-seq数据几乎相同,这证实了转录组数据的准确性和可靠性。此外,我们还在稻瘟病感染的d17、d14和WT植物的叶片中进行了H2O2和可溶性糖测量的组织化学检测。为了支持转录组数据,这里的生化结果显示,相对于野生型水稻叶片,两种受损突变体(d17和d14)的叶片中H2O2的积累(由DAB染色强度证明)和可溶性糖含量显着降低,进一步指向SLs促进H2O2和糖合成的作用,特别是在生物胁迫过程中。此外,我们还研究了合成SLs(GR5)对稻瘟病菌生长的直接影响。观察到SLs(GR5)对稻瘟病的生长无显著影响,这进一步支持了植物内SLs的介导防御机制,而不是其对稻瘟病生长的直接影响。总而言之,这些发现表明SL-生物合成和信号通过参与细胞壁,乙烯,H2O2和糖生物合成相关基因/途径的诱导而促进对稻瘟病感染的防御。该研究的结果表明,SLs可能是增强水稻对稻瘟病菌防御能力的新靶点。进一步研究,为了解决SLs生物合成和信号传导是否会影响水稻根际微生物以及如何决定水稻根际微生物的问题,我们分析并比较了d17突变体,d14突变体及野生型水稻(WT)的根际微生物群(基于细菌的16S rRNA序列和真菌的ITS序列)。与WT相比,SLs生物合成的d17突变体和d14信号传导突变体的根际中观察到更高的细菌丰富度(OTU丰富度)和更低的真菌多样性(Shannon指数)。此外,相对于WT,d14的细菌多样性(辛普森指数)极显著降低,而在d17中显著降低。基于Bray-Curtis距离的方差的主坐标分析和置换多元分析显示,与WT相比,d17和d14的根际微生物的群落组成具有显著差异。值得关注的是与野生型水稻相比,d17和d14突变体中大量细菌群落组成上存在显着差异,而存在差异的真菌群落较少。这表明水稻SLs生物合成和信号传导对构建细菌群落产生了更大的影响,对真菌群落产生较少的影响。重要的是,16s rRNA测序表明,相对于WT,两种突变基因型中某些有益细菌分类群(包括亚硝化单胞菌科、和产黄杆菌)的相对丰度显着降低。亚硝化单胞菌科和芽单胞菌门的成员可能分别促进硝化和耐旱,而产黄杆菌中的物种能作为潜在的生物抑制剂。该发现提供了间接证据,即SLs生物合成和信号可能通过相应细菌组合物的正调节介导各种生物和生物胁迫能力。此外,为了理解SLs依赖性代谢途径如何影响水稻中的根际微生物,进行了植物KEGG代谢途径相关基因(基于转录组数据)和根际微生物群落组成之间的相关性分析。结果表明,富集的KEGG代谢途径相关特异基因与WT的根际微生物正相关。在这方面,特定的SLs依赖性代谢途径,包括苯丙氨酸代谢、苯丙醇生物合成、植物激素信号转导途径过氧化物酶体、乙醛酸和二羧酸代谢、半乳糖代谢和丙氨酸、天门冬氨酸和谷氨酸代谢与野生型富集的不同微生物群落类群呈正相关。这些发现表明,SLs生物合成和信号传导通过直接或间接影响不同代谢途径来决定水稻根际微生物群落的组成。总之,这些结果表明SLs生物合成和信号传导不仅可以正向调节水稻对稻瘟病菌的防御,而且在群落结构中具有十分重要的作用,尤其是根际细菌群落,通过直接或间接参与不同代谢途径的调节来影响根际微生物的群落组成。基于研究结果,我们提出通过转基因方法对SLs生物合成或信号传导途径的遗传调控,可以提供一种新的策略来增强水稻对稻瘟病菌的防御机制,并且可以帮助富集所需且有益根际微生物,这可能有助于水稻或其他作物的抗压能力。进而,这将使我们能够实现确保可持续粮食安全的最终目标。
Abstract
shui dao (Oryza sativa L.)zuo wei zhu yao de liang shi zuo wu zhi yi ,man zu le shi jie ren kou yao yi ban de liang shi xu qiu 。yu ji dao 2050nian ,quan qiu ren kou jiang zeng chang dao yao 90yi 。yin ci ,wei le man zu zhe yi zeng chang xu qiu ,di gao shui dao chan liang zhi guan chong yao 。ran er ,you yu dao wen bing jun (Magnaporthe oryzae)yin qi de dao wen bing dao zhi quan qiu mei nian shui dao chan liang sun shi yan chong (yao 10-30%),quan qiu liang shi an quan wen ti yi ran shou dao wei xie 。tong chang shi yong bu tong de sha jun ji lai kong zhi dao wen bing ,li ru san huan zuo 、bing xi zuo 、mi jun zhi 、bing huan zuo he yi bing chun 。zhe xie sha jun ji sui ran zhi bing lv gao 、jian xiao kuai ,dan shi hua xue sha jun ji de bu kuo dang huo guo du shi yong ,ke neng dui ren lei jian kang zao cheng qian zai wei ji ,wei xie sheng tai huan jing 。li yong ju you “chong zu te yi xing ”kang bing de zuo wu pin chong shi ling yi chong ke kao de ji bing zhi li fang fa 。dan shi ,you yu bu duan ke fu su zhu fang yu de dao wen bing xin xing bing yuan ti de chu xian ,kang bing xing bing bu chi jiu ,zhi neng chi xu liang san nian zuo you 。yin ci ,pai qie xu yao neng gou di gao shui dao dui dao wen bing jun mian yi li de fang fa yi huo de dui dao wen bing geng chi jiu de kang xing 。wei le di yu ge chong ge yang de sheng wu he fei sheng wu xie pai ,zhi wu bu jin ji huo ji chu fang yu fan ying ,bao gua fang yu ji su de chan sheng ,hai yi lai yu gen ji wei sheng wu de sheng cun he kuo ying neng li 。gen ji wei sheng wu tong guo can yu su zhu yang fen huo qu he huan jing ya li hui fu ,shi ji cheng wei yi sheng tai ke chi xu fang shi di gao nong ye shou yi de li xiang fang fa 。you qu de shi ,te ding de zhi wu ji su he xiang guan de xin hao bu jin zuo wei zhi wu sheng chang he fang yu diao jie yin zi ,er ju zai shua ze te ding de gen ji wei sheng wu cheng fen fang mian fa hui zhao ji ji de zuo yong 。yin ci ,tan suo zhi wu ji su zai fang yu zhong de zuo yong ,que ding gen ji wei sheng wu ji ji xiang guan ji zhi ,jiang wei sheng tai ke chi xu fang shi di gao zuo wu chan liang di gong xin de tu jing 。du jiao jin nei zhi (SLs)shi yi lei zhi wu ji su ,zai zhi wu fa yo he sheng li guo cheng de diao jie zhong qi guan jian zuo yong 。ci wai ,xin de zheng ju biao ming ,SLshai ke yi cu jin zhi wu de te ding sheng wu he fei sheng wu kang xing 。SLshai chong dang gen ji xin hao yin zi ;cong er ji ji diao jie ji zhu zhi wu yu cong zhi jun gen zhen jun zhi jian de gong sheng guan ji 。ci wai ,zui jin hai bao dao le SLsdui gen ji wei sheng wu qun la zu cheng ju you diao jie zuo yong 。zhe xie fa xian biao ming le zhi wu yu wei sheng wu xiang hu zuo yong guo cheng zhong SLsde qian li 。ran er ,ju wo men suo zhi ,shui dao zai dao wen bing jun de ying xiang xia yu gen ji wei sheng wu qun la zhi jian xiang hu zuo yong guo cheng zhong ,SLssheng wu ge cheng he xin hao de ke neng diao jie zuo yong he ji zhi shang bu qing chu 。yin ci ,zai zhe xiang yan jiu zhong ,wo men yan jiu le shui dao zai dao wen bing jun de ying xiang xia yu gen ji wei sheng wu qun la zhi jian xiang hu zuo yong guo cheng zhong SLssheng wu ge cheng he xin hao de diao jie zuo yong 。shou xian ,wo men tong guo SLssheng wu ge cheng huo xin hao chuan dao zhong shou sun de shui dao tu bian ti ji ji xiang ying de ye sheng xing (WT;Oryza sativa L.cv.Shiokari)de bi jiao fen xi ,zai dao wen bing jun gan ran shui dao zhi wu de guo cheng zhong ,tan tao le SLssheng wu ge cheng he xin hao zai fang yu dao wen bing zhong de zuo yong 。jie guo biao ming ,ru guo SLssheng wu ge cheng tu bian ti (dwarf17,d17)huo xin hao chuan dao tu bian ti (d14)hui dao zhi shui dao dui dao wen bing jun de yi gan xing zeng jia 。zhe qing chu de biao ming SLssheng wu ge cheng he xin hao chuan dao neng gou di gao dui dao wen bing de kang xing 。wei le di gao shui dao dui dao wen bing jun de kang xing ,tan tao le SLsdiao jie de xia you ji yin /tu jing ,dui d14tu bian ti he ye sheng xing shui dao zhi zhu (you huo mo M.oryzaegan ran )jin hang le quan ji yin zu bi jiao xie zhuai lu zu xue (ji yu Illuminagao tong liang ce xu de RNAce xu )fen xi 。ran hou ,shi yong MapManfen xi zhuai lu zu shu ju bing huo de sheng wu xie pai tu jing 。ji yu MapMande zhuai lu zu shu ju ji de sheng wu xie pai xian shi ,zai shui dao dao wen bing jun gan ran ji jian ,d14tu bian ti xie pian zhong xiang dang shu liang de fang yu xiang guan ji yin (bao gua xi bao bi 、yi xi 、guo yang hua qing ge cheng xiang guan ji yin )xiang dui yu ye sheng zhi wu ming xian shou dao yi zhi 。ci wai ,KEGGfu ji fen xi biao ming ,yu ye sheng xing shui dao xiang bi ,d14tu bian ti zhong de dai xie tu jing zhu yao she ji tan shui hua ge wu /tang dai xie ,bao gua dian fen he zhe tang dai xie 、wu tang lin suan tu jing 、yi quan suan he er suo suan dai xie yi ji guang ge zuo yong zai dao wen bing gan ran ji jian bei xian zhe yi zhi 。tong guo fen xi yu yi xi sheng wu ge cheng ,H2O2sheng wu ge cheng he tan shui hua ge wu ge cheng tu jing xiang guan de 8ge ji yin de biao da mo shi yi ji shi yong ying guang ding liang PCR(qRT-PCR)de yan zheng 7ge sui ji shua ze de ji yin lai yan zheng zhuai lu zu shu ju jie guo 。zong de lai shui ,qRT-PCRhuo de de jie guo yu RNA-seqshu ju ji hu xiang tong ,zhe zheng shi le zhuai lu zu shu ju de zhun que xing he ke kao xing 。ci wai ,wo men hai zai dao wen bing gan ran de d17、d14he WTzhi wu de xie pian zhong jin hang le H2O2he ke rong xing tang ce liang de zu zhi hua xue jian ce 。wei le zhi chi zhuai lu zu shu ju ,zhe li de sheng hua jie guo xian shi ,xiang dui yu ye sheng xing shui dao xie pian ,liang chong shou sun tu bian ti (d17he d14)de xie pian zhong H2O2de ji lei (you DABran se jiang du zheng ming )he ke rong xing tang han liang xian zhao jiang di ,jin yi bu zhi xiang SLscu jin H2O2he tang ge cheng de zuo yong ,te bie shi zai sheng wu xie pai guo cheng zhong 。ci wai ,wo men hai yan jiu le ge cheng SLs(GR5)dui dao wen bing jun sheng chang de zhi jie ying xiang 。guan cha dao SLs(GR5)dui dao wen bing de sheng chang mo xian zhe ying xiang ,zhe jin yi bu zhi chi le zhi wu nei SLsde jie dao fang yu ji zhi ,er bu shi ji dui dao wen bing sheng chang de zhi jie ying xiang 。zong er yan zhi ,zhe xie fa xian biao ming SL-sheng wu ge cheng he xin hao tong guo can yu xi bao bi ,yi xi ,H2O2he tang sheng wu ge cheng xiang guan ji yin /tu jing de you dao er cu jin dui dao wen bing gan ran de fang yu 。gai yan jiu de jie guo biao ming ,SLske neng shi zeng jiang shui dao dui dao wen bing jun fang yu neng li de xin ba dian 。jin yi bu yan jiu ,wei le jie jue SLssheng wu ge cheng he xin hao chuan dao shi fou hui ying xiang shui dao gen ji wei sheng wu yi ji ru he jue ding shui dao gen ji wei sheng wu de wen ti ,wo men fen xi bing bi jiao le d17tu bian ti ,d14tu bian ti ji ye sheng xing shui dao (WT)de gen ji wei sheng wu qun (ji yu xi jun de 16S rRNAxu lie he zhen jun de ITSxu lie )。yu WTxiang bi ,SLssheng wu ge cheng de d17tu bian ti he d14xin hao chuan dao tu bian ti de gen ji zhong guan cha dao geng gao de xi jun feng fu du (OTUfeng fu du )he geng di de zhen jun duo yang xing (Shannonzhi shu )。ci wai ,xiang dui yu WT,d14de xi jun duo yang xing (xin pu sen zhi shu )ji xian zhe jiang di ,er zai d17zhong xian zhe jiang di 。ji yu Bray-Curtisju li de fang cha de zhu zuo biao fen xi he zhi huan duo yuan fen xi xian shi ,yu WTxiang bi ,d17he d14de gen ji wei sheng wu de qun la zu cheng ju you xian zhe cha yi 。zhi de guan zhu de shi yu ye sheng xing shui dao xiang bi ,d17he d14tu bian ti zhong da liang xi jun qun la zu cheng shang cun zai xian zhao cha yi ,er cun zai cha yi de zhen jun qun la jiao shao 。zhe biao ming shui dao SLssheng wu ge cheng he xin hao chuan dao dui gou jian xi jun qun la chan sheng le geng da de ying xiang ,dui zhen jun qun la chan sheng jiao shao de ying xiang 。chong yao de shi ,16s rRNAce xu biao ming ,xiang dui yu WT,liang chong tu bian ji yin xing zhong mou xie you yi xi jun fen lei qun (bao gua ya xiao hua chan bao jun ke 、he chan huang gan jun )de xiang dui feng du xian zhao jiang di 。ya xiao hua chan bao jun ke he ya chan bao jun men de cheng yuan ke neng fen bie cu jin xiao hua he nai han ,er chan huang gan jun zhong de wu chong neng zuo wei qian zai de sheng wu yi zhi ji 。gai fa xian di gong le jian jie zheng ju ,ji SLssheng wu ge cheng he xin hao ke neng tong guo xiang ying xi jun zu ge wu de zheng diao jie jie dao ge chong sheng wu he sheng wu xie pai neng li 。ci wai ,wei le li jie SLsyi lai xing dai xie tu jing ru he ying xiang shui dao zhong de gen ji wei sheng wu ,jin hang le zhi wu KEGGdai xie tu jing xiang guan ji yin (ji yu zhuai lu zu shu ju )he gen ji wei sheng wu qun la zu cheng zhi jian de xiang guan xing fen xi 。jie guo biao ming ,fu ji de KEGGdai xie tu jing xiang guan te yi ji yin yu WTde gen ji wei sheng wu zheng xiang guan 。zai zhe fang mian ,te ding de SLsyi lai xing dai xie tu jing ,bao gua ben bing an suan dai xie 、ben bing chun sheng wu ge cheng 、zhi wu ji su xin hao zhuai dao tu jing guo yang hua wu mei ti 、yi quan suan he er suo suan dai xie 、ban ru tang dai xie he bing an suan 、tian men dong an suan he gu an suan dai xie yu ye sheng xing fu ji de bu tong wei sheng wu qun la lei qun cheng zheng xiang guan 。zhe xie fa xian biao ming ,SLssheng wu ge cheng he xin hao chuan dao tong guo zhi jie huo jian jie ying xiang bu tong dai xie tu jing lai jue ding shui dao gen ji wei sheng wu qun la de zu cheng 。zong zhi ,zhe xie jie guo biao ming SLssheng wu ge cheng he xin hao chuan dao bu jin ke yi zheng xiang diao jie shui dao dui dao wen bing jun de fang yu ,er ju zai qun la jie gou zhong ju you shi fen chong yao de zuo yong ,you ji shi gen ji xi jun qun la ,tong guo zhi jie huo jian jie can yu bu tong dai xie tu jing de diao jie lai ying xiang gen ji wei sheng wu de qun la zu cheng 。ji yu yan jiu jie guo ,wo men di chu tong guo zhuai ji yin fang fa dui SLssheng wu ge cheng huo xin hao chuan dao tu jing de wei chuan diao kong ,ke yi di gong yi chong xin de ce lve lai zeng jiang shui dao dui dao wen bing jun de fang yu ji zhi ,bing ju ke yi bang zhu fu ji suo xu ju you yi gen ji wei sheng wu ,zhe ke neng you zhu yu shui dao huo ji ta zuo wu de kang ya neng li 。jin er ,zhe jiang shi wo men neng gou shi xian que bao ke chi xu liang shi an quan de zui zhong mu biao 。
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