【作者】 李翔；

【导师】 张明方；

【作者基本信息】 浙江大学 ， 蔬菜学， 2019， 博士

【摘要】 线粒体到细胞核的信号传递通常被称作线粒体反向调控(Mitochondrial Retrograde Regulation,MRR),在植物、动物与微生物中普遍存在。高等植物线粒体反向调控信号受多种胁迫诱导,如各种生物胁迫与非生物胁迫,以及线粒体呼吸抑制剂(抗霉素A等)。核编码线粒体定位的交替氧化酶基因(Alternative oxidasela,AOX1a),被认为是植物线粒体反向信号调控的标记基因,能被抗霉素A诱导表达。近些年对模式植物拟南芥线粒体反向调控的研究中,鉴定到线粒体反向调控信号的一些关键因子,比如AtMYB29,AtNAC013。芥菜细胞质雄性不育系CMS是典型的线粒体DNA突变体,可以与其保持系MF、自然育性回复突变系REV系统作为试验材料来研究线粒体反向调控信号途径。实验室前期研究发现,拟南芥中与酵母14-3-3基因Bmh1/2高度同源的AtGRF3在线粒体反向调控中作为负调控因子起重要作用。此外,我们发现一些14-3-3基因在芥菜REV/CMS转录谱中存在显著表达差异。因此,本课题旨在研究芥菜14-3-3基因在线粒体反向调控信号途径中的作用,利用VIGS对候选基因进行沉默以验证其功能,通过酵母双杂研究其作用机制。主要研究结果如下:1.细胞质雄性不育系中线粒体反向调控信号途径发生改变在抗霉素A胁迫下,芥菜CMS系中AOX1a的诱导表达显著低于MF与REV,表明芥菜CMS中的线粒体反向调控信号途径受到一定程度的抑制。在白菜CMS系,萝卜CMS系与烟草CMS系中AOX1a的诱导表达也显著低于其MF,从而进一步验证了这一结论。2.线粒体反向调控途径候选基因BjuGRF2、BjuGRF3的鉴定与筛选本研究以拟南芥14-3-3基因为参照,根据序列同源性与功能结构域保守性鉴定了芥菜、白菜与黑芥中的14-3-3基因家族,其中芥菜37个基因,白菜21个基因,黑芥26个基因。通过对芥菜MF、CMS与REV系进行基因表达分析,我们发现BjuGRF2(BjuB026023)与BjuGRF3(BjuA026371)在CMS系中表达均显著高于MF与REV。用抗霉素A处理后,BjuGRF2在CMS系中表达下调,而在MF与REV中上调表达;BjuGRF3在CMS系中表达无显著差异,但是在MF与REV中上调表达。3.芥菜14-3-3家族基因BjGRF2、BjGRF3在线粒体反向调控中起负调控作用分别构建芥菜BjuGRF2以及BjuGRF3的VIGS载体,侵染植株后成功获得BjuGRF2与BjuGRF3的沉默株系。在BjuGRF2沉默株系中,AOX1a的表达高于对照植株中的表达;通过使用AA处理沉默株系与对照植株,并进行基因表达分析,我们发现在沉默株系中,AOX1a的诱导表达显著高于对照植株,这一结果表明BjuGRF2负调控线粒体反向调控信号途径。对BjuGRF3沉默株系与对照植株进行AA处理与基因表达分析,我们发现沉默株系的AOX1a诱导表达显著高于对照植株,这一结果表明BjuGRF3也是AOX1a的负调控因子,在线粒体反向调控信号途径起负调控作用。4.芥菜 14-3-3 蛋白(BjuGRF2、BjuGRF3)与转录因子(BjuMYB29、BjuNAC013)蛋白互作通过酵母双杂实验,我们发现BjuGRF2蛋白与BjuMYB29蛋白互作,BjuGRF3蛋白与BjuNAC013互作,据此推测BjuGRF2、BjuGRF3是通过与线粒体反向调控信号途径转录因子的互作来调控AOX1a的基因表达,在线粒体反向调控中起负调控作用。更多还原

【Abstract】 The signal transduction from mitochondrion to the nucleus is usually named Mitochondrial retrograde regulation(MRR),generally exsiting in plant,animal and eukaryote.Mitochondrial retrograde regulation can be induced by variety stress,including biotic stress,abiotic stress and mitochondrial respiration inhibitors(antimycin A).Alternative oxidasela(AOX1a)is a nuclear gene encoding mitochondrial proten,which is regarded as a report gene of mitochondrial retrograde regulation and can be induced by antimycin A(AA).The study of Arabidopsis th.aliana recently showed that transcription factors(TFs)play important role in MRR.For example,AtMYB29 AtNAC013.Cytoplasmic Male Sterility(CMS)of Brassica juncea is mtDNA Mutant.we can use it to study Mitochondrial reverse signal with its maintenance(MF)and revertant(REV).Formerly,we found that AtGRF3 is a homologue of 14-3-3 gene Bmhl/2 in yeast,and AtGRF3 is a negative regulator of Mitochondrial retrograde regulation.Furthermore,we found some 14-3-3 genes exist difference in REV/CMS expression profile of Brassica juncea.Thus,our research aim at study the function of 14-3-3 genes in Mitochondrial reverse signal,taking advantage of VIGS to silence and verificate the function of 14-3-3 genes and by the way of yeast two hybrid to study the interaction between 14-3-3 proteins and TFs.The main results are as follows:1.Mitochondrial reverse signaling pathway changes in CMS plantsCompared with MF and REV plants,the expression level of AOXla in CMS plants is lower significantly under antimycin A treatment.Moreover,the induced expression of AOXla in Brassica rapa CMS&MF.Rapha1nus Sativus CMS&MF and Nicotiana tabacum CMS&MF was similar to that in Brassica juncea.These results verified that Mitochondrial reverse signaling pathway changes in CMS plants.2.Identification and screening of candidate genes(BjuGRF2,BjuGRF3)in Mitochondrial reverse signaling pathway of Brassica junceaWe identified the 14-3-3 genes in Brassica juncea,Brassica rapa and Brassica nigra based on the sequence homology and structure domains referring to Arabidopsis thaliana,and in total we identified 37 genes in Brassica juncea,21 genes in Brassica rapa and 26 genes in Brassica nigra.Expression profiles of 14-3-3 genes in Brassica juncea MF,CMS and REV plants were analysed using qRT-PCR.The results indicate that BjuGRF2(BjuB026023)and BjuGRF3(BjuA026371)is highly expressed in CMS plants than in MF and REV plants.Under the treatment with AA,we found that the expression of BjuGRF1 in CMS plants is lower than control,while the expression of BjuGRF2 in MF and REV plants is higher than control;at the same time,it shows no difference of BjuGRF3 expression between AA treatment and control in CMS plant,but the expression of BjuGRF3 in MF and REV plants is higher than control3.BjuGRF2 and BjuGRF3 is negative regulator of Mitochondrial retrograde regulation in Brassica junceaWe constructed the VIGS vector of BjuGRF2,BjuGRF3 seperately and successfully silenced the gene expression of BjuGRF2 and BjuGRF3.Under antimycin A treatment.the expression of AOXla in BjuGRF2 silenced plants is induced remarkebly,but the induced expression of AOX1a is lower in Mock plants,which indicate that BjuGRF2 negativly regulates MRR.The induced expression of AOX1a in BjuGRF3 silenced plants is higher than Mock plants with AA treatment,it reveals that BjuGRF3 negativly regulates MRR.4.14-3-3 protein(BjuGRF2,BjuGRF3)interacts with transcription factors in Brassica junceaYeast two hybrid results showed that BjuGRF1 interacts with BjuMYB29.and BjuGRF3 interactw with BjuNAC013.This may sugested that 14-3-3 proteins through the interaction with transcription factors negatively regulate the expression of AOXla in Mitochondrial reverse signal pathway.更多还原