利用VIGS技术进行油菜抗病相关WRKY70和LRR-RLK基因的功能验证

发布时间：2018-02-04 17:01

  本文关键词： 油菜 VIGS WRKY70 LRR-RLK　出处：《内蒙古大学》2017年硕士论文　论文类型：学位论文

【摘要】：WRKY70和LRR-RLK在许多植物中都参与了植物的抗病反应,WRKY70是植物信号传导过程中的重要成员,在植物抗病过程中发挥着重要作用,主要参与了茉莉酸(JA)与水杨酸(SA)途径,LRR-RLK可以直接或间接的识别病原物,并将信号传递下去,使植物产生一系列抗病反应。本研究在利用黑胫病菌侵染油菜的转录组测序基础上,筛选高表达及差异表达基因,结合油菜全基因组测序数据,获得WRKY70和LRR-RLK两个基因全长,为了深入了解这两个基因的信息及验证其在油菜中的抗病功能,为鉴定油菜抗病通路中重要基因的功能提供数据信息,利用生物信息学进行了序列分析,然后采用VIGS技术,将携带外源基因的cDNA导入宿主,诱发宿主同源基因沉默,来达到验证基因功能的目的。取得的研究结果如下:1.根据前期用弱侵染型菌株NM-1侵染油菜得到的转录组测序结果中WRK Y70和LRR-RLK基因的全长CDS序列,利用CLC SequenceViewer 6推导WR KY70和LRR-RLK氨基酸序列,并对WRKY70和LRR-RLK氨基酸序列进行蛋白大小、等电点、结构域、磷酸化位点、N-糖基化位点以及疏水性分析。其中W RKY70基因CDS全长858bp,编码285个氨基酸,含有一个WRKY保守结构域,属于Ⅲ类WRKY转录因子;LRR-RLK基因CDS全长2775bp,编码924个氨基酸,含有丝氨酸苏氨酸蛋白激酶活性位点。2.克隆WRKY70和LRR-RLK基因部分CDS序列,WRKY70片段大小为308bp,LRR-RLK基因片段大小为394bp,测序后进行序列比对,与转录组序列100%一致。3.将成功构建的VIGS沉默载体pTRV2-WRKY70和pTRV2-LRR-RLK,转入大肠杆菌MC1061中进行菌液PCR及单、双酶切验证后,提取质粒转化农杆菌GV3101,用于侵染油菜叶片。4.农杆菌介导的沉默载体侵染油菜叶片14d后接种黑胫病病原菌NM-1,收集4h、12h、24h、36h、48h、96h叶片,提取RNA通过qPCR检测目的基因表达量,并观察植株发病情况。结果显示,沉默植株中目的基因的相对表达量低于未沉默植株,并且沉默植株叶片的病斑较大,叶片褪绿发黄较明显。
[Abstract]:WRKY70 and LRR-RLK are important members of plant signal transduction and play an important role in plant disease resistance. LRR-RLK can directly or indirectly identify the pathogen and transmit the signal through the pathway of JAA (jasmonic acid) and SAA (salicylate). On the basis of the transcriptome sequencing of Brassica napus, high expression and differentially expressed genes were screened, combined with the whole genome sequencing data of Brassica napus. Two full-length WRKY70 and LRR-RLK genes were obtained in order to understand the information of the two genes and to verify their resistance function in rape. In order to provide data information for identifying the function of important genes in resistance pathway of rapeseed, sequence analysis was carried out by bioinformatics, and then cDNA carrying exogenous gene was introduced into host by VIGS technique. Induce homologous gene silencing. The results are as follows: 1.According to the results of transcriptome sequencing of Brassica napus infected with weakly infecting strain NM-1. The full-length CDS sequence of Y70 and LRR-RLK gene. The amino acid sequences of WR KY70 and LRR-RLK were deduced by CLC SequenceViewer 6. The amino acid sequences of WRKY70 and LRR-RLK were analyzed for protein size, isoelectric point, domain and phosphorylation site. The N-glycosylation site and hydrophobicity analysis showed that the CDS of WRKY 70 gene was 858 BP in length, encoding 285 amino acids and containing a conserved domain of WRKY. It belongs to class 鈪，

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