摘要
利用接头PCR技术首次分离了长度为1 216bp 的“双优”山葡萄(Vitis amurensis Rupr.) classIII几丁质酶基因VCH3上游启动子序列(GenBank登录号AF441123),并用引物延伸方法鉴定了该启动子的转录起始位点,为5'-ATCAAGCAC-3'序列中的第二个A。序列分析结果表明,代表真核基因启动子特征的CAAT 盒和TATA 盒分别位于VCH3启动子转录起始位点上游?122和?29处。另外,在转录起始位点上游?1 181 bp 和?293 bp 处各有一个水杨酸(SA)响应的顺式作用元件TGACG。为了鉴定该启动子的功能,将该启动子连接到β-葡糖苷酸酶基因(GUS)编码区的上游构建了VCH3启动子-GUS融合基因,并用农杆菌介导叶盘转化法将该融合基因转入烟草栽培品种NC89 中。SA处理的转基因烟草根系和叶片GUS酶活性的荧光和组织化学检测结果表明VCH3启动子的驱动作用被SA诱导,因而该启动子在基因工程中将具有潜在的应用价值。
The 1 216-bp 5＇ upstream region of the gene encoding a class Ⅲ chitinase VCH3 was isolated from grapevine （Vitis amurensis Rupr.） by adaptor-PCR （GenBank accession number AF441123）, and the transcriptional start site of the VCH3 gene was identified by primer extension, which corresponds to the second A in the DNA sequence 5＇-ATCAAGCAC-3＇. Sequence analysis revealed that the VCH3 promoter sequence contains CAAT and TATA motifs that are located at the -122 and -29 nucleotide upstream of the transcriptional start site, respectively. Both motifs are characteristic of the eukaryotic gene promoter. In addition, within the VCH3 promoter, we found two inverse salicylic acid （SA）-responsive cis-acting motifs （TGACG） at -1 181 bp and -293 bp upstream of the transcriptional start site, respectively. To characterize the VCH3 promoter, the VCH3 promoter-GUS chimera was constructed and transferred to Nicotiana tobacum cv. NC89 by Agrobacterium tumefaciensmediated leaf disc transformation. Fluorometric and histochemical analysis of GUS activity in the transgenic tobacco root and leaf treated with SA showed that the VCH3 promoter was induced by SA, which indicates that it may have potential use in genetic engineering.
出处
《植物生理与分子生物学学报》
CAS
CSCD
北大核心
2005年第5期485-491,共7页
Journal Of Plant Physiology and Molecular Biology
基金
国家自然科学基金青年基金项目(No. 30400300)
吉林省杰出青年基金项目(No. 20050117)
吉林省转基因专项(No. 20040209-2)
吉林省科技发展计划项目(No. 20030553-1)资助~~
