本文主要研究内容
作者刘笑玮（2019）在《PVY6K2蛋白与本氏烟RubisCO小亚基蛋白的互作研究》一文中研究指出：马铃薯Y病毒(Potato virus Y,PVY)是一种正义单链RNA病毒,寄主范围广泛,能侵染马铃薯、辣椒、番茄等植物,严重影响农产品的质量和产量,给生产带来巨大的经济损失。研究PVY与寄主互作关系,有助于深入揭示PVY的致病机理,为马铃薯Y病毒病的防治提供理论依据。PVY编码的膜蛋白6K2能诱导形成含有病毒复制复合体(VRC)的复制囊泡,对病毒复制有着不可或缺的调控作用。笔者所在实验室前期利用PVY 6K2蛋白为“诱饵”,通过酵母双杂交技术(Y2H)对模式植物本氏烟(Nicotiana benthamiana)cDNA文库进行筛选,筛选出13种可能与PVY 6K2蛋白存在互作的蛋白质。本研究以筛选到的N.benthamiana中疑似互作蛋白1,5-二磷酸核酮糖羧化酶/加氧酶(RubisCO)的小亚基蛋白(NbRbcS)为研究对象,利用分子生物学、生物化学、细胞生物学和病毒生物学等技术手段,验证PVY 6K2蛋白与寄主NbRbcS蛋白存在互作,明确寄主NbRbcS蛋白对PVY侵染复制的调控作用,主要研究结果如下:(1)PVY 6K2蛋白与寄主NbRbcS蛋白互作。为验证筛库结果,通过同源克隆技术,克隆了NbRbcS基因,获得了NbRbcS基因序列,并进行了系统进化分析。同时,通过酵母双杂交(Y2H)和免疫共沉淀(Co-IP)实验,分别从体外及体内验证了PVY 6K2蛋白的确能与寄主NbRbcS蛋白互作。(2)PVY 6K2蛋白能改变NbRbcS蛋白的亚细胞定位,并降低RubisCO酶的活性及叶绿素含量。为明确PVY侵染对寄主NbRbcS蛋白的调控,利用荧光定量PCR(real-time RT-PCR)进行验证,发现PVY侵染后,NbRbcS基因mRNA水平呈现先上升后下降的趋势,暗示NbRbcS蛋白可能参与了寄主抗PVY的免疫防卫反应。构建RbcS-RFP融合蛋白,激光共聚焦显微镜观察发现无论有无PVY侵染,6K2蛋白浸润均可改变NbRbcS蛋白的定位,将其从叶绿体招募至内质网附近。通过分光光度法分别对RubisCO酶活及叶绿素的含量进行测定,发现PVY侵染及6K2蛋白浸润时,均可抑制RubisCO酶的活性及叶绿素的合成。(3)NbRbcS蛋白对PVY侵染有抑制作用。为进一步验证寄主NbRbcS蛋白对PVY侵染复制的影响,利用病毒诱导的基因沉默技术(VIGS)将寄主NbRbcS基因沉默,结果发现NbRbcS基因沉默后,会引起本氏烟叶片严重的褪绿症状,及类似于PVY初侵染的明脉症状,推测PVY初侵染造成的明脉症状与NbRbcS基因表达下调和功能丧失有关。接种PVY后,发现寄主NbRbcS基因沉默促进PVY的侵染复制,局部叶片过表达NbRbcS基因验证了NbRbcS蛋白对PVY侵染有抑制作用。基于以上研究结果总结如下:PVY侵染本氏烟时,可利用自身编码的膜蛋白6K2与寄主NbRbcS蛋白互作,改变NbRbcS蛋白在细胞内的定位,将其从叶绿体招募到内质网上,并抑制NbRbcS蛋白的表达,进而影响RubisCO酶的活性及叶绿素的合成,从而促进病毒侵染,产生明脉症状。该研究结果进一步加深了对6K2蛋白在PVY复制和侵染中作用的了解,揭示了PVY侵染初期明脉症状产生的分子机制,为农业上防治马铃薯Y病毒病及抗病毒药物的研发提供理论依据。
Abstract
ma ling shu Ybing du (Potato virus Y,PVY)shi yi chong zheng yi chan lian RNAbing du ,ji zhu fan wei an fan ,neng qin ran ma ling shu 、la jiao 、fan jia deng zhi wu ,yan chong ying xiang nong chan pin de zhi liang he chan liang ,gei sheng chan dai lai ju da de jing ji sun shi 。yan jiu PVYyu ji zhu hu zuo guan ji ,you zhu yu shen ru jie shi PVYde zhi bing ji li ,wei ma ling shu Ybing du bing de fang zhi di gong li lun yi ju 。PVYbian ma de mo dan bai 6K2neng you dao xing cheng han you bing du fu zhi fu ge ti (VRC)de fu zhi nang pao ,dui bing du fu zhi you zhao bu ke huo que de diao kong zuo yong 。bi zhe suo zai shi yan shi qian ji li yong PVY 6K2dan bai wei “you er ”,tong guo jiao mu shuang za jiao ji shu (Y2H)dui mo shi zhi wu ben shi yan (Nicotiana benthamiana)cDNAwen ku jin hang shai shua ,shai shua chu 13chong ke neng yu PVY 6K2dan bai cun zai hu zuo de dan bai zhi 。ben yan jiu yi shai shua dao de N.benthamianazhong yi shi hu zuo dan bai 1,5-er lin suan he tong tang suo hua mei /jia yang mei (RubisCO)de xiao ya ji dan bai (NbRbcS)wei yan jiu dui xiang ,li yong fen zi sheng wu xue 、sheng wu hua xue 、xi bao sheng wu xue he bing du sheng wu xue deng ji shu shou duan ,yan zheng PVY 6K2dan bai yu ji zhu NbRbcSdan bai cun zai hu zuo ,ming que ji zhu NbRbcSdan bai dui PVYqin ran fu zhi de diao kong zuo yong ,zhu yao yan jiu jie guo ru xia :(1)PVY 6K2dan bai yu ji zhu NbRbcSdan bai hu zuo 。wei yan zheng shai ku jie guo ,tong guo tong yuan ke long ji shu ,ke long le NbRbcSji yin ,huo de le NbRbcSji yin xu lie ,bing jin hang le ji tong jin hua fen xi 。tong shi ,tong guo jiao mu shuang za jiao (Y2H)he mian yi gong chen dian (Co-IP)shi yan ,fen bie cong ti wai ji ti nei yan zheng le PVY 6K2dan bai de que neng yu ji zhu NbRbcSdan bai hu zuo 。(2)PVY 6K2dan bai neng gai bian NbRbcSdan bai de ya xi bao ding wei ,bing jiang di RubisCOmei de huo xing ji xie lu su han liang 。wei ming que PVYqin ran dui ji zhu NbRbcSdan bai de diao kong ,li yong ying guang ding liang PCR(real-time RT-PCR)jin hang yan zheng ,fa xian PVYqin ran hou ,NbRbcSji yin mRNAshui ping cheng xian xian shang sheng hou xia jiang de qu shi ,an shi NbRbcSdan bai ke neng can yu le ji zhu kang PVYde mian yi fang wei fan ying 。gou jian RbcS-RFProng ge dan bai ,ji guang gong ju jiao xian wei jing guan cha fa xian mo lun you mo PVYqin ran ,6K2dan bai jin run jun ke gai bian NbRbcSdan bai de ding wei ,jiang ji cong xie lu ti qiao mu zhi nei zhi wang fu jin 。tong guo fen guang guang du fa fen bie dui RubisCOmei huo ji xie lu su de han liang jin hang ce ding ,fa xian PVYqin ran ji 6K2dan bai jin run shi ,jun ke yi zhi RubisCOmei de huo xing ji xie lu su de ge cheng 。(3)NbRbcSdan bai dui PVYqin ran you yi zhi zuo yong 。wei jin yi bu yan zheng ji zhu NbRbcSdan bai dui PVYqin ran fu zhi de ying xiang ,li yong bing du you dao de ji yin chen mo ji shu (VIGS)jiang ji zhu NbRbcSji yin chen mo ,jie guo fa xian NbRbcSji yin chen mo hou ,hui yin qi ben shi yan xie pian yan chong de tui lu zheng zhuang ,ji lei shi yu PVYchu qin ran de ming mai zheng zhuang ,tui ce PVYchu qin ran zao cheng de ming mai zheng zhuang yu NbRbcSji yin biao da xia diao he gong neng sang shi you guan 。jie chong PVYhou ,fa xian ji zhu NbRbcSji yin chen mo cu jin PVYde qin ran fu zhi ,ju bu xie pian guo biao da NbRbcSji yin yan zheng le NbRbcSdan bai dui PVYqin ran you yi zhi zuo yong 。ji yu yi shang yan jiu jie guo zong jie ru xia :PVYqin ran ben shi yan shi ,ke li yong zi shen bian ma de mo dan bai 6K2yu ji zhu NbRbcSdan bai hu zuo ,gai bian NbRbcSdan bai zai xi bao nei de ding wei ,jiang ji cong xie lu ti qiao mu dao nei zhi wang shang ,bing yi zhi NbRbcSdan bai de biao da ,jin er ying xiang RubisCOmei de huo xing ji xie lu su de ge cheng ,cong er cu jin bing du qin ran ,chan sheng ming mai zheng zhuang 。gai yan jiu jie guo jin yi bu jia shen le dui 6K2dan bai zai PVYfu zhi he qin ran zhong zuo yong de le jie ,jie shi le PVYqin ran chu ji ming mai zheng zhuang chan sheng de fen zi ji zhi ,wei nong ye shang fang zhi ma ling shu Ybing du bing ji kang bing du yao wu de yan fa di gong li lun yi ju 。
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论文详细介绍
论文作者分别是来自中国农业科学院的刘笑玮，发表于刊物中国农业科学院2019-07-05论文，是一篇关于植物病毒互作论文,马铃薯病毒论文,蛋白论文,蛋白论文,中国农业科学院2019-07-05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，资料来自中国农业科学院2019-07-05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：植物病毒互作论文; 马铃薯病毒论文; 蛋白论文; 中国农业科学院2019-07-05论文;