本文主要研究内容
作者顾少涵（2019）在《番茄PUB基因家族在灰霉病抗性形成中的作用研究》一文中研究指出：农作物暴露在细菌、真菌和病毒等病原微生物环境中,极易受到病虫害的侵害。保护地内温暖湿润的环境导致设施栽培作物病害尤为严重。番茄灰霉病作为一种典型的真菌病害给番茄的产量及质量造成了严重的危害。目前,生产上高度依赖化学杀菌剂,给生态环境和食品安全造成严重威胁。通过基因编辑技术CRISPR/Cas9可以实现灰霉病的精准调控以及抗灰霉病种质创新。因此,迫切需要探究作物防御灰霉病的内在机制。蛋白质泛素化是真核生物重要的翻译后修饰方式之一,在多种信号转导途径中发挥作用。近年来的研究表明,泛素化广泛地参与植物免疫防御调节。PUB(Plant U-box)家族是一类含有U-box结构域的E3泛素连接酶(Ubiquitin-ligating enzyme,E3),可以通过一系列泛素化修饰的级联反应,作为关键元件参与免疫防御。本文通过对番茄PUB基因家族生物信息学分析,进一步筛选出负调控灰霉病抗性的番茄PUB基因家族成员,对于明确植物防御灰霉病的内在机制具有重要意义。主要研究成果如下:1.番茄PUB基因家族生物信息学分析。通过与拟南芥PUB基因家族成员氨基酸序列进行比对,找到番茄中共含有U-box结构域的61个PUB编码基因。除了保守U-box结构域外,还存在ARM(Armadillo)重复序列,激酶等其他结构域。根据结构域的不同,将61个番茄PUB基因分成七大类。其中,数量最多的是含有ARM重复结构的第II类,共30个。ARM重复结构通过蛋白互作从而调控信号转导,所以我们推测番茄PUB第II类基因在植物免疫防御功能中具有重要意义。2.筛选出负调控灰霉病抗性的番茄PUB家族成员。通过上一章对番茄PUB基因家族进行生物信息学分析,推测番茄PUB第II类基因在植物免疫调控中具有重要意义。通过对番茄PUB家族第II类基因进行荧光定量PCR(Quantitive Real-Time PCR,qRT-PCR)分析,初步筛选出对灰霉病响应明显下调的8个基因PCB15/24/25/29/33/41/43/56。通过烟草瞬时过表达加接种灰霉病菌的实验以及共注射过表达PUBs与灰霉分泌蛋白XYG农杆菌菌液的方式,进一步筛选出负调控番茄灰霉病抗性的番茄PUB家族成员PUB33、PUB41、PUB43以及PUB56。将筛选出的基因进行病毒诱导基因沉默(Virus-induced gene silencing,VIGS)。实验结果表明,沉默PUB33、PUB41、PUB43以及PUB56之后,植株对于灰霉病的抗性提高。通过CRISPR/Cas9技术成功构建PUB41突变体材料,pub41对灰霉病的抗性也明显提高,表明这些基因对于番茄防御灰霉病具有重要意义。3.初步探究了番茄PUB基因负调控番茄灰霉病抗性的机理。上一章已经筛选出负调控番茄灰霉病抗性的番茄PUB家族成员。已有文章表明,PSKR1以及TPK1b是番茄防御灰霉病的关键因子。用CRISPR/Cas9技术获得PSKR1以及TPK1b的突变体材料,接种灰霉病菌后发现,pskr1以及tpk1b与对照植株相比对灰霉病的抗性下降,说明PSKR1以及TPK1b正调控番茄灰霉病抗性。除此之外,我们还发现PUB33定位于细胞膜上,PUB43以及PUB56定位于细胞膜及细胞质内。并且PUB43以及PUB56具有E3泛素连接酶功能。通过双分子荧光互补(Biomolecular fluorescence complementation,BIFC)实验证明 PUB43 与 PSKR1以及TPK1b之间存在互作。初步认为PUB43可能通过泛素化降解PSKR1以及TPK1b,从而引起下游信号传递。
Abstract
nong zuo wu bao lou zai xi jun 、zhen jun he bing du deng bing yuan wei sheng wu huan jing zhong ,ji yi shou dao bing chong hai de qin hai 。bao hu de nei wen nuan shi run de huan jing dao zhi she shi zai pei zuo wu bing hai you wei yan chong 。fan jia hui mei bing zuo wei yi chong dian xing de zhen jun bing hai gei fan jia de chan liang ji zhi liang zao cheng le yan chong de wei hai 。mu qian ,sheng chan shang gao du yi lai hua xue sha jun ji ,gei sheng tai huan jing he shi pin an quan zao cheng yan chong wei xie 。tong guo ji yin bian ji ji shu CRISPR/Cas9ke yi shi xian hui mei bing de jing zhun diao kong yi ji kang hui mei bing chong zhi chuang xin 。yin ci ,pai qie xu yao tan jiu zuo wu fang yu hui mei bing de nei zai ji zhi 。dan bai zhi fan su hua shi zhen he sheng wu chong yao de fan yi hou xiu shi fang shi zhi yi ,zai duo chong xin hao zhuai dao tu jing zhong fa hui zuo yong 。jin nian lai de yan jiu biao ming ,fan su hua an fan de can yu zhi wu mian yi fang yu diao jie 。PUB(Plant U-box)jia zu shi yi lei han you U-boxjie gou yu de E3fan su lian jie mei (Ubiquitin-ligating enzyme,E3),ke yi tong guo yi ji lie fan su hua xiu shi de ji lian fan ying ,zuo wei guan jian yuan jian can yu mian yi fang yu 。ben wen tong guo dui fan jia PUBji yin jia zu sheng wu xin xi xue fen xi ,jin yi bu shai shua chu fu diao kong hui mei bing kang xing de fan jia PUBji yin jia zu cheng yuan ,dui yu ming que zhi wu fang yu hui mei bing de nei zai ji zhi ju you chong yao yi yi 。zhu yao yan jiu cheng guo ru xia :1.fan jia PUBji yin jia zu sheng wu xin xi xue fen xi 。tong guo yu ni na gai PUBji yin jia zu cheng yuan an ji suan xu lie jin hang bi dui ,zhao dao fan jia zhong gong han you U-boxjie gou yu de 61ge PUBbian ma ji yin 。chu le bao shou U-boxjie gou yu wai ,hai cun zai ARM(Armadillo)chong fu xu lie ,ji mei deng ji ta jie gou yu 。gen ju jie gou yu de bu tong ,jiang 61ge fan jia PUBji yin fen cheng qi da lei 。ji zhong ,shu liang zui duo de shi han you ARMchong fu jie gou de di IIlei ,gong 30ge 。ARMchong fu jie gou tong guo dan bai hu zuo cong er diao kong xin hao zhuai dao ,suo yi wo men tui ce fan jia PUBdi IIlei ji yin zai zhi wu mian yi fang yu gong neng zhong ju you chong yao yi yi 。2.shai shua chu fu diao kong hui mei bing kang xing de fan jia PUBjia zu cheng yuan 。tong guo shang yi zhang dui fan jia PUBji yin jia zu jin hang sheng wu xin xi xue fen xi ,tui ce fan jia PUBdi IIlei ji yin zai zhi wu mian yi diao kong zhong ju you chong yao yi yi 。tong guo dui fan jia PUBjia zu di IIlei ji yin jin hang ying guang ding liang PCR(Quantitive Real-Time PCR,qRT-PCR)fen xi ,chu bu shai shua chu dui hui mei bing xiang ying ming xian xia diao de 8ge ji yin PCB15/24/25/29/33/41/43/56。tong guo yan cao shun shi guo biao da jia jie chong hui mei bing jun de shi yan yi ji gong zhu she guo biao da PUBsyu hui mei fen bi dan bai XYGnong gan jun jun ye de fang shi ,jin yi bu shai shua chu fu diao kong fan jia hui mei bing kang xing de fan jia PUBjia zu cheng yuan PUB33、PUB41、PUB43yi ji PUB56。jiang shai shua chu de ji yin jin hang bing du you dao ji yin chen mo (Virus-induced gene silencing,VIGS)。shi yan jie guo biao ming ,chen mo PUB33、PUB41、PUB43yi ji PUB56zhi hou ,zhi zhu dui yu hui mei bing de kang xing di gao 。tong guo CRISPR/Cas9ji shu cheng gong gou jian PUB41tu bian ti cai liao ,pub41dui hui mei bing de kang xing ye ming xian di gao ,biao ming zhe xie ji yin dui yu fan jia fang yu hui mei bing ju you chong yao yi yi 。3.chu bu tan jiu le fan jia PUBji yin fu diao kong fan jia hui mei bing kang xing de ji li 。shang yi zhang yi jing shai shua chu fu diao kong fan jia hui mei bing kang xing de fan jia PUBjia zu cheng yuan 。yi you wen zhang biao ming ,PSKR1yi ji TPK1bshi fan jia fang yu hui mei bing de guan jian yin zi 。yong CRISPR/Cas9ji shu huo de PSKR1yi ji TPK1bde tu bian ti cai liao ,jie chong hui mei bing jun hou fa xian ,pskr1yi ji tpk1byu dui zhao zhi zhu xiang bi dui hui mei bing de kang xing xia jiang ,shui ming PSKR1yi ji TPK1bzheng diao kong fan jia hui mei bing kang xing 。chu ci zhi wai ,wo men hai fa xian PUB33ding wei yu xi bao mo shang ,PUB43yi ji PUB56ding wei yu xi bao mo ji xi bao zhi nei 。bing ju PUB43yi ji PUB56ju you E3fan su lian jie mei gong neng 。tong guo shuang fen zi ying guang hu bu (Biomolecular fluorescence complementation,BIFC)shi yan zheng ming PUB43 yu PSKR1yi ji TPK1bzhi jian cun zai hu zuo 。chu bu ren wei PUB43ke neng tong guo fan su hua jiang jie PSKR1yi ji TPK1b,cong er yin qi xia you xin hao chuan di 。
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论文详细介绍
论文作者分别是来自浙江大学的顾少涵，发表于刊物浙江大学2019-05-20论文，是一篇关于番茄论文,灰霉病论文,泛素化论文,互作论文,浙江大学2019-05-20论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自浙江大学2019-05-20论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：番茄论文; 灰霉病论文; 泛素化论文; 互作论文; 浙江大学2019-05-20论文;