本文主要研究内容
作者马世伟（2019）在《水稻抗稻瘟病新基因的功能分析和Gα参与抗稻瘟病机理研究》一文中研究指出：水稻(Oryza sativa L.)是最重要的粮食作物之一,全球超过1/2人口以稻米为主食。由稻瘟病菌(Magnaporthe grisea)引起的稻瘟病可导致水稻减产,严重时甚至颗粒无收。常见防治稻瘟病的方法主要有应用杀菌剂、农艺措施以及培育抗性品种。由于杀虫剂的大量使用使环境受到严重污染。因此,培育和推广广谱抗病水稻新品种是最经济、最安全、最有效的稻瘟病防治手段。但稻瘟病菌菌株变异快,常常导致新推广的抗性品种在应用数年后抗性降低或者抗性丧失。因此,不断发掘广谱抗性基因,揭示水稻抗稻瘟病的分子机制,为稻瘟病抗性品种选育提供理论指导,对水稻的安全生产具有重要意义。本研究以水稻品种CO39及其对稻瘟病抗、感能力不同的5个近等基因系C101LAC、C101A51、C104PKT、C101PKT和C105TTP-4L-23(简称Pi1、Pi2、Pi3、Pi4a和Pi4b)为材料,在前期对供试水稻品种基因组重测序和稻瘟病菌胁迫下供试水稻转录组测序的基础上,应用组学比较技术分别从基因组重测序和转录组测序数据中各筛选获得潜在的水稻抗稻瘟病基因,以抗高毒力的稻瘟病菌GUY11的水稻品种Pi4b为材料,应用CRISPR/Cas9技术构建2个上述筛选获得的潜在抗稻瘟病基因的突变体,明确基因的抗稻瘟病功能,并分析其作用机理。另外,本研究也构建了水稻异三聚体G蛋白α亚基的突变体d1-Pi4b,阐明了Gα蛋白在水稻抗稻瘟病防御反应过程的信号转导机制。具体研究结果如下。1.揭示OsBRG1基因介导水稻抗稻瘟病及调控籽粒发育的机理从水稻品种CO39的5个近等基因系基因组重测序与比较基因组学分析结果中遴选出潜在抗稻瘟病基因OsBRG1,首先应用CRISPR/Cas9技术构建OsBRG1基因突变体,比较稻瘟病菌GUY11侵染7d后OsBRG1基因突变体及其野生型的叶片表型,发现OsBRG1基因突变体叶片发病明显,为感病4级,野生型Pi4b为1级抗病,研究结果验证表明OsBRG1是一个水稻抗稻瘟病的新基因。然后,分析该基因的基本特性,发现OsBRG1基因编码一个NBS-LRR蛋白,具有典型的保守结构域NBS和LRR,亚细胞定位结果显示该蛋白定位于细胞质和细胞膜,超敏反应结果进一步确定了该基因的抗稻瘟病菌GUY11的功能。最后,分析了OsBRG1基因抗稻瘟病的机理。OsBRG1基因能够响应植物激素SA、JA、ET和ABA处理,表明该基因能通过4种激素途径参与水稻应对生物与非生物逆境胁迫的响应过程。在稻瘟病菌GUY11侵染下,抗、感稻瘟病水稻品种中该基因均被上调表达,验证了该基因能响应生物逆境胁迫-稻瘟菌侵染胁迫。高温诱导OsBRG1基因的上调表达,同样验证了该基因能参与水稻应对非生物逆境胁迫的生理响应。再分析OsBRG1基因突变体中抗病相关基因的表达模式,发现稻瘟菌侵染下抗病相关基因MAPK6、WRKY45、PR1a、PR2、PR3和PR10均被上调表达,且在野生型Pi4b中的表达量显著高于OsBRG1突变体,表明OsBRG1基因通过协同促进抗病相关基因的表达介导了水稻的抗稻瘟病能力。同时,水稻原生质体免疫沉淀试验从OsBRG1蛋白质的复合体中分离获得59个蛋白质,经过酵母双杂交验证了其中一个蛋白质Os07g0105900能与OsBRG1蛋白质互作,且Os07g0105900也是一个抗病基因,表明Os07g0105900和OsBRG1基因协同介导了水稻抗稻瘟病作用。比较OsBRG1基因的M-1、M-2型突变体与野生型籽粒,发现M-1型突变体的籽粒长度大于M-2型和野生型Pi4b的,其中M-2型与野生型无差异;M-1型与M-2型的籽粒宽度相同,均小于野生型;M-1型的千粒重为25.68 g,比野生型增加了10.0%,且M-1型突变体籽粒长宽比最大、胚乳透明度最高、垩白粒率最低,结果表明OsBRG1基因能调控水稻籽粒的形态发育。分析OsBRG1基因M-1、M-2型突变体和野生型水稻品种幼穗发育各个阶段已知籽粒相关基因的表达模式,发现在幼穗发育至2.0、3.0、9.0和16.0 cm时,在M-1型突变体中籽粒发育基因GL7的表达量显著高于M-2型和野生型,在籽粒发育到2.0 cm时两者间的差异最大,M-1型的是野生型的4.8倍,说明OsBRG1基因通过调控GL7基因的表达而影响水稻籽粒的发育。2.揭示OsBRW1基因介导水稻抗稻瘟病的机理从稻瘟病菌胁迫下近等基因系水稻品种Pi1、Pi4a和Pi4b的转录组测序数据中,经比较转录组学分析,遴选出水稻潜在抗稻瘟病基因OsBRW1,进行基因的功能验证与作用机理研究。首先应用CRISPR/Cas9技术构建OsBRW1基因突变体,比较稻瘟病菌GUY11侵染7d后OsBRW1基因突变体及其野生型的叶片表型,发现OsBRW1基因突变体叶片发病明显,为感病4级,野生型Pi4b为1级抗病,研究结果确定了OsBRW1是一个水稻抗稻瘟病的新基因。然后,分析该基因的基本特性,发现OsBRW1基因编码NBS-ARC蛋白质,具有典型的保守结构域NBS和CC。亚细胞定位结果显示该蛋白定位于细胞质和细胞核,超敏反应结果进一步验证了该基因抗稻瘟病菌GUY11的功能。最后,分析了OsBRW1基因抗稻瘟病的机理。OsBRW1基因能够响应植物激素SA、JA、ET和ABA处理,表明该基因能通过4种激素途径参与水稻应对生物和非生物逆境胁迫的生理响应。在稻瘟病菌GUY11侵染下,抗、感稻瘟病水稻品种中该基因均被上调表达,进一步验证了该基因能响应生物逆境胁迫-稻瘟菌侵染胁迫。分析OsBRW1基因突变体抗病相关基因的表达模式,发现稻瘟菌侵染下抗病相关基因MAPK6/12、WRKY45、PR1a、PR2、PR3和PR10均被上调表达,且野生型Pi4b的表达量显著高于OsBRW1突变体的,结果表明OsBRW1基因通过促进抗病相关基因的协同表达介导了水稻的抗稻瘟病作用。3.明确了异三聚体G蛋白α亚基参与水稻抗稻瘟病响应的机理首先应用qPCR分析GUY11侵染下,CO39及其4个近等基因系水稻品种中Gα的表达模式,发现Gα基因在CO39中下调表达,在其余各品种中均持续上调表达,表明Gα基因可能参与水稻抗稻瘟病的反应过程。然后应用CRISPR/Cas9技术构建Gα基因突变体d1-Pi4b,比较稻瘟病菌GUY11侵染7 d后d1-Pi4b突变体及其野生型的叶片表型,发现d1-Pi4b叶片发病明显,发病等级为3级,野生型Pi4b为1级抗病,进一步明确了Gα能参与水稻抗稻瘟病的反应过程。最后,利用原核表达系统,表达并纯化Gα蛋白,以纯化后的蛋白质为抗原免疫家兔制备Gα蛋白的多克隆抗血清,收集GUY11侵染下品种Pi4b叶片并以抗血清进行IP试验,经质谱分析Gα蛋白质复合体中的112个蛋白质,经BiFC验证其中的Hsp70能与Gα直接互作。结果表明,Gα蛋白通过与Hsp70互作组成蛋白质复合体,在细胞内外级联传递稻瘟病菌胁迫信号,继而引发水稻植株的分子应答,产生抗稻瘟病的防御反应。总之,本文挖掘并验证了两个水稻新抗稻瘟病基因OsBRG1和OsBRW1,并初步揭示了其抗病的分子机制,且发现OsBRG1基因能够调控水稻籽粒的形态发育。此外,本研究也明确了异三聚体G蛋白α亚基能参与水稻抗稻瘟病的生理响应。研究结果为抗稻瘟病水稻新品种的选育奠定了理论基础。
Abstract
shui dao (Oryza sativa L.)shi zui chong yao de liang shi zuo wu zhi yi ,quan qiu chao guo 1/2ren kou yi dao mi wei zhu shi 。you dao wen bing jun (Magnaporthe grisea)yin qi de dao wen bing ke dao zhi shui dao jian chan ,yan chong shi shen zhi ke li mo shou 。chang jian fang zhi dao wen bing de fang fa zhu yao you ying yong sha jun ji 、nong yi cuo shi yi ji pei yo kang xing pin chong 。you yu sha chong ji de da liang shi yong shi huan jing shou dao yan chong wu ran 。yin ci ,pei yo he tui an an pu kang bing shui dao xin pin chong shi zui jing ji 、zui an quan 、zui you xiao de dao wen bing fang zhi shou duan 。dan dao wen bing jun jun zhu bian yi kuai ,chang chang dao zhi xin tui an de kang xing pin chong zai ying yong shu nian hou kang xing jiang di huo zhe kang xing sang shi 。yin ci ,bu duan fa jue an pu kang xing ji yin ,jie shi shui dao kang dao wen bing de fen zi ji zhi ,wei dao wen bing kang xing pin chong shua yo di gong li lun zhi dao ,dui shui dao de an quan sheng chan ju you chong yao yi yi 。ben yan jiu yi shui dao pin chong CO39ji ji dui dao wen bing kang 、gan neng li bu tong de 5ge jin deng ji yin ji C101LAC、C101A51、C104PKT、C101PKThe C105TTP-4L-23(jian chen Pi1、Pi2、Pi3、Pi4ahe Pi4b)wei cai liao ,zai qian ji dui gong shi shui dao pin chong ji yin zu chong ce xu he dao wen bing jun xie pai xia gong shi shui dao zhuai lu zu ce xu de ji chu shang ,ying yong zu xue bi jiao ji shu fen bie cong ji yin zu chong ce xu he zhuai lu zu ce xu shu ju zhong ge shai shua huo de qian zai de shui dao kang dao wen bing ji yin ,yi kang gao du li de dao wen bing jun GUY11de shui dao pin chong Pi4bwei cai liao ,ying yong CRISPR/Cas9ji shu gou jian 2ge shang shu shai shua huo de de qian zai kang dao wen bing ji yin de tu bian ti ,ming que ji yin de kang dao wen bing gong neng ,bing fen xi ji zuo yong ji li 。ling wai ,ben yan jiu ye gou jian le shui dao yi san ju ti Gdan bai αya ji de tu bian ti d1-Pi4b,chan ming le Gαdan bai zai shui dao kang dao wen bing fang yu fan ying guo cheng de xin hao zhuai dao ji zhi 。ju ti yan jiu jie guo ru xia 。1.jie shi OsBRG1ji yin jie dao shui dao kang dao wen bing ji diao kong zi li fa yo de ji li cong shui dao pin chong CO39de 5ge jin deng ji yin ji ji yin zu chong ce xu yu bi jiao ji yin zu xue fen xi jie guo zhong lin shua chu qian zai kang dao wen bing ji yin OsBRG1,shou xian ying yong CRISPR/Cas9ji shu gou jian OsBRG1ji yin tu bian ti ,bi jiao dao wen bing jun GUY11qin ran 7dhou OsBRG1ji yin tu bian ti ji ji ye sheng xing de xie pian biao xing ,fa xian OsBRG1ji yin tu bian ti xie pian fa bing ming xian ,wei gan bing 4ji ,ye sheng xing Pi4bwei 1ji kang bing ,yan jiu jie guo yan zheng biao ming OsBRG1shi yi ge shui dao kang dao wen bing de xin ji yin 。ran hou ,fen xi gai ji yin de ji ben te xing ,fa xian OsBRG1ji yin bian ma yi ge NBS-LRRdan bai ,ju you dian xing de bao shou jie gou yu NBShe LRR,ya xi bao ding wei jie guo xian shi gai dan bai ding wei yu xi bao zhi he xi bao mo ,chao min fan ying jie guo jin yi bu que ding le gai ji yin de kang dao wen bing jun GUY11de gong neng 。zui hou ,fen xi le OsBRG1ji yin kang dao wen bing de ji li 。OsBRG1ji yin neng gou xiang ying zhi wu ji su SA、JA、EThe ABAchu li ,biao ming gai ji yin neng tong guo 4chong ji su tu jing can yu shui dao ying dui sheng wu yu fei sheng wu ni jing xie pai de xiang ying guo cheng 。zai dao wen bing jun GUY11qin ran xia ,kang 、gan dao wen bing shui dao pin chong zhong gai ji yin jun bei shang diao biao da ,yan zheng le gai ji yin neng xiang ying sheng wu ni jing xie pai -dao wen jun qin ran xie pai 。gao wen you dao OsBRG1ji yin de shang diao biao da ,tong yang yan zheng le gai ji yin neng can yu shui dao ying dui fei sheng wu ni jing xie pai de sheng li xiang ying 。zai fen xi OsBRG1ji yin tu bian ti zhong kang bing xiang guan ji yin de biao da mo shi ,fa xian dao wen jun qin ran xia kang bing xiang guan ji yin MAPK6、WRKY45、PR1a、PR2、PR3he PR10jun bei shang diao biao da ,ju zai ye sheng xing Pi4bzhong de biao da liang xian zhe gao yu OsBRG1tu bian ti ,biao ming OsBRG1ji yin tong guo xie tong cu jin kang bing xiang guan ji yin de biao da jie dao le shui dao de kang dao wen bing neng li 。tong shi ,shui dao yuan sheng zhi ti mian yi chen dian shi yan cong OsBRG1dan bai zhi de fu ge ti zhong fen li huo de 59ge dan bai zhi ,jing guo jiao mu shuang za jiao yan zheng le ji zhong yi ge dan bai zhi Os07g0105900neng yu OsBRG1dan bai zhi hu zuo ,ju Os07g0105900ye shi yi ge kang bing ji yin ,biao ming Os07g0105900he OsBRG1ji yin xie tong jie dao le shui dao kang dao wen bing zuo yong 。bi jiao OsBRG1ji yin de M-1、M-2xing tu bian ti yu ye sheng xing zi li ,fa xian M-1xing tu bian ti de zi li chang du da yu M-2xing he ye sheng xing Pi4bde ,ji zhong M-2xing yu ye sheng xing mo cha yi ;M-1xing yu M-2xing de zi li kuan du xiang tong ,jun xiao yu ye sheng xing ;M-1xing de qian li chong wei 25.68 g,bi ye sheng xing zeng jia le 10.0%,ju M-1xing tu bian ti zi li chang kuan bi zui da 、pei ru tou ming du zui gao 、e bai li lv zui di ,jie guo biao ming OsBRG1ji yin neng diao kong shui dao zi li de xing tai fa yo 。fen xi OsBRG1ji yin M-1、M-2xing tu bian ti he ye sheng xing shui dao pin chong you sui fa yo ge ge jie duan yi zhi zi li xiang guan ji yin de biao da mo shi ,fa xian zai you sui fa yo zhi 2.0、3.0、9.0he 16.0 cmshi ,zai M-1xing tu bian ti zhong zi li fa yo ji yin GL7de biao da liang xian zhe gao yu M-2xing he ye sheng xing ,zai zi li fa yo dao 2.0 cmshi liang zhe jian de cha yi zui da ,M-1xing de shi ye sheng xing de 4.8bei ,shui ming OsBRG1ji yin tong guo diao kong GL7ji yin de biao da er ying xiang shui dao zi li de fa yo 。2.jie shi OsBRW1ji yin jie dao shui dao kang dao wen bing de ji li cong dao wen bing jun xie pai xia jin deng ji yin ji shui dao pin chong Pi1、Pi4ahe Pi4bde zhuai lu zu ce xu shu ju zhong ,jing bi jiao zhuai lu zu xue fen xi ,lin shua chu shui dao qian zai kang dao wen bing ji yin OsBRW1,jin hang ji yin de gong neng yan zheng yu zuo yong ji li yan jiu 。shou xian ying yong CRISPR/Cas9ji shu gou jian OsBRW1ji yin tu bian ti ,bi jiao dao wen bing jun GUY11qin ran 7dhou OsBRW1ji yin tu bian ti ji ji ye sheng xing de xie pian biao xing ,fa xian OsBRW1ji yin tu bian ti xie pian fa bing ming xian ,wei gan bing 4ji ,ye sheng xing Pi4bwei 1ji kang bing ,yan jiu jie guo que ding le OsBRW1shi yi ge shui dao kang dao wen bing de xin ji yin 。ran hou ,fen xi gai ji yin de ji ben te xing ,fa xian OsBRW1ji yin bian ma NBS-ARCdan bai zhi ,ju you dian xing de bao shou jie gou yu NBShe CC。ya xi bao ding wei jie guo xian shi gai dan bai ding wei yu xi bao zhi he xi bao he ,chao min fan ying jie guo jin yi bu yan zheng le gai ji yin kang dao wen bing jun GUY11de gong neng 。zui hou ,fen xi le OsBRW1ji yin kang dao wen bing de ji li 。OsBRW1ji yin neng gou xiang ying zhi wu ji su SA、JA、EThe ABAchu li ,biao ming gai ji yin neng tong guo 4chong ji su tu jing can yu shui dao ying dui sheng wu he fei sheng wu ni jing xie pai de sheng li xiang ying 。zai dao wen bing jun GUY11qin ran xia ,kang 、gan dao wen bing shui dao pin chong zhong gai ji yin jun bei shang diao biao da ,jin yi bu yan zheng le gai ji yin neng xiang ying sheng wu ni jing xie pai -dao wen jun qin ran xie pai 。fen xi OsBRW1ji yin tu bian ti kang bing xiang guan ji yin de biao da mo shi ,fa xian dao wen jun qin ran xia kang bing xiang guan ji yin MAPK6/12、WRKY45、PR1a、PR2、PR3he PR10jun bei shang diao biao da ,ju ye sheng xing Pi4bde biao da liang xian zhe gao yu OsBRW1tu bian ti de ,jie guo biao ming OsBRW1ji yin tong guo cu jin kang bing xiang guan ji yin de xie tong biao da jie dao le shui dao de kang dao wen bing zuo yong 。3.ming que le yi san ju ti Gdan bai αya ji can yu shui dao kang dao wen bing xiang ying de ji li shou xian ying yong qPCRfen xi GUY11qin ran xia ,CO39ji ji 4ge jin deng ji yin ji shui dao pin chong zhong Gαde biao da mo shi ,fa xian Gαji yin zai CO39zhong xia diao biao da ,zai ji yu ge pin chong zhong jun chi xu shang diao biao da ,biao ming Gαji yin ke neng can yu shui dao kang dao wen bing de fan ying guo cheng 。ran hou ying yong CRISPR/Cas9ji shu gou jian Gαji yin tu bian ti d1-Pi4b,bi jiao dao wen bing jun GUY11qin ran 7 dhou d1-Pi4btu bian ti ji ji ye sheng xing de xie pian biao xing ,fa xian d1-Pi4bxie pian fa bing ming xian ,fa bing deng ji wei 3ji ,ye sheng xing Pi4bwei 1ji kang bing ,jin yi bu ming que le Gαneng can yu shui dao kang dao wen bing de fan ying guo cheng 。zui hou ,li yong yuan he biao da ji tong ,biao da bing chun hua Gαdan bai ,yi chun hua hou de dan bai zhi wei kang yuan mian yi jia tu zhi bei Gαdan bai de duo ke long kang xie qing ,shou ji GUY11qin ran xia pin chong Pi4bxie pian bing yi kang xie qing jin hang IPshi yan ,jing zhi pu fen xi Gαdan bai zhi fu ge ti zhong de 112ge dan bai zhi ,jing BiFCyan zheng ji zhong de Hsp70neng yu Gαzhi jie hu zuo 。jie guo biao ming ,Gαdan bai tong guo yu Hsp70hu zuo zu cheng dan bai zhi fu ge ti ,zai xi bao nei wai ji lian chuan di dao wen bing jun xie pai xin hao ,ji er yin fa shui dao zhi zhu de fen zi ying da ,chan sheng kang dao wen bing de fang yu fan ying 。zong zhi ,ben wen wa jue bing yan zheng le liang ge shui dao xin kang dao wen bing ji yin OsBRG1he OsBRW1,bing chu bu jie shi le ji kang bing de fen zi ji zhi ,ju fa xian OsBRG1ji yin neng gou diao kong shui dao zi li de xing tai fa yo 。ci wai ,ben yan jiu ye ming que le yi san ju ti Gdan bai αya ji neng can yu shui dao kang dao wen bing de sheng li xiang ying 。yan jiu jie guo wei kang dao wen bing shui dao xin pin chong de shua yo dian ding le li lun ji chu 。
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标签：水稻论文; 突变体论文; 抗稻瘟病新基因论文; 亚基论文; 分子机制论文; 福建农林大学2019-08-30论文;