本文關(guān)鍵詞： 油菜 VIGS WRKY70 LRR-RLK　出處：《內(nèi)蒙古大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：WRKY70和LRR-RLK在許多植物中都參與了植物的抗病反應(yīng),WRKY70是植物信號(hào)傳導(dǎo)過程中的重要成員,在植物抗病過程中發(fā)揮著重要作用,主要參與了茉莉酸(JA)與水楊酸(SA)途徑,LRR-RLK可以直接或間接的識(shí)別病原物,并將信號(hào)傳遞下去,使植物產(chǎn)生一系列抗病反應(yīng)。本研究在利用黑脛病菌侵染油菜的轉(zhuǎn)錄組測(cè)序基礎(chǔ)上,篩選高表達(dá)及差異表達(dá)基因,結(jié)合油菜全基因組測(cè)序數(shù)據(jù),獲得WRKY70和LRR-RLK兩個(gè)基因全長(zhǎng),為了深入了解這兩個(gè)基因的信息及驗(yàn)證其在油菜中的抗病功能,為鑒定油菜抗病通路中重要基因的功能提供數(shù)據(jù)信息,利用生物信息學(xué)進(jìn)行了序列分析,然后采用VIGS技術(shù),將攜帶外源基因的cDNA導(dǎo)入宿主,誘發(fā)宿主同源基因沉默,來達(dá)到驗(yàn)證基因功能的目的。取得的研究結(jié)果如下:1.根據(jù)前期用弱侵染型菌株NM-1侵染油菜得到的轉(zhuǎn)錄組測(cè)序結(jié)果中WRK Y70和LRR-RLK基因的全長(zhǎng)CDS序列,利用CLC SequenceViewer 6推導(dǎo)WR KY70和LRR-RLK氨基酸序列,并對(duì)WRKY70和LRR-RLK氨基酸序列進(jìn)行蛋白大小、等電點(diǎn)、結(jié)構(gòu)域、磷酸化位點(diǎn)、N-糖基化位點(diǎn)以及疏水性分析。其中W RKY70基因CDS全長(zhǎng)858bp,編碼285個(gè)氨基酸,含有一個(gè)WRKY保守結(jié)構(gòu)域,屬于Ⅲ類WRKY轉(zhuǎn)錄因子;LRR-RLK基因CDS全長(zhǎng)2775bp,編碼924個(gè)氨基酸,含有絲氨酸蘇氨酸蛋白激酶活性位點(diǎn)。2.克隆WRKY70和LRR-RLK基因部分CDS序列,WRKY70片段大小為308bp,LRR-RLK基因片段大小為394bp,測(cè)序后進(jìn)行序列比對(duì),與轉(zhuǎn)錄組序列100%一致。3.將成功構(gòu)建的VIGS沉默載體pTRV2-WRKY70和pTRV2-LRR-RLK,轉(zhuǎn)入大腸桿菌MC1061中進(jìn)行菌液PCR及單、雙酶切驗(yàn)證后,提取質(zhì)粒轉(zhuǎn)化農(nóng)桿菌GV3101,用于侵染油菜葉片。4.農(nóng)桿菌介導(dǎo)的沉默載體侵染油菜葉片14d后接種黑脛病病原菌NM-1,收集4h、12h、24h、36h、48h、96h葉片,提取RNA通過qPCR檢測(cè)目的基因表達(dá)量,并觀察植株發(fā)病情況。結(jié)果顯示,沉默植株中目的基因的相對(duì)表達(dá)量低于未沉默植株,并且沉默植株葉片的病斑較大,葉片褪綠發(fā)黃較明顯。
[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，

本文編號(hào)：1490699