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油菜菌核病菌激发子基因SsXYN克隆及表达载体构建
蒋冰心,吴群,高智谋,张华建
0
(安徽农业大学植物保护学院,合肥 230036)
摘要:
以核盘菌菌株NGA4提取的总RNA为模版,利用RT-PCR技术扩增获得SsXYN基因的全长cDNA片段,将其克隆到pMD19-T载体上,菌落PCR、酶切鉴定和cDNA测序结果表明成功克隆了核盘菌SsXYN基因。从pMD19-T : SsXYN载体上,用BamH I和Sal I 双酶切切下目的基因片段,将该基因片段连接到原核表达载体pET32a中。菌落PCR和酶切鉴定结果显示成功构建了表达载体pET32a : SsXYN。利用热击法将该重组表达载体导入大肠杆菌BL21,获得携带SsXYN基因的大肠杆菌菌株,为进一步研究激发子诱发植物抗病性机理奠定了基础。
关键词:  核盘菌  SsXYN  激发子
DOI:
投稿时间:2013-03-28
基金项目:国家自然科学青年基金项目(31101426)和公益性行业(农业)科研专项(201103016)共同资助。
Cloning of Sclerotinia sclerotiorum elicitor gene SsXYN and the expression vector construction
JIANG Bing-xin,WU Qun,GAO Zhi-mou,ZHANG Hua-jian
(School of Plant Protection, Anhui Agricultural University, Hefei 230036)
Abstract:
Total RNA was extracted from Sclerotinia sclerotiorum NGA4 mycelia and used as the template to amplify the full length of cDNA of SsXYN gene by RT-PCR, and the gene fragment was subsequently cloned into pMD19-T vector. The results of bacterial colony PCR, enzyme analysis and sequencing analysis indicated that the S. sclerotiorum SsXYN gene was successfully cloned. SsXYN gene was digested completely from pMD19-T : SsXYN by BamH I and Sal I, and then was cloned into expression vector pET32a. The results of bacterial colony PCR and enzyme analysis showed the expression vector pET32a : SsXYN was successfully constructed. After that, the recombinant expression vector was carried into E.coli BL21 by heat-shock transformation, and the strain of E.coli BL21 carrying SsXYN gene was obtained. The study provides a basis for our understanding of the elicitor SsXYN downstream signaling pathway in plant immunity.
Key words:  Sclerotinia sclerotiorum  SsXYN  elicitor

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