一个新的马铃薯patatin class Ⅰ基因的cDNA在烟草中的表达及其酶活性  

Expression of a Novel Potato Patatin Class Ⅰ cDNA in Transgenic Tobacco and Its Lipid Acylhydrolase Activity

作　　者：司怀军[1] 柳俊[2] 谢从华[1] 

机构地区：[1]华中农业大学园艺林学学院,武汉430070 [2]华中农业大学生命科学技术学院,武汉430070

出　　处：《农业生物技术学报》2003年第3期236-240,共5页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金资助项目(39970464)。

摘　　要：将马铃薯(Solanumtuberosum )块茎特异蛋白patatinclassⅠ基因cDNA与CaMV35S启动子融合,构建了植物表达载体pBSSP。用电激法将表达载体导入根癌农杆菌(Agrobacteriumtumefaciens)LBA4404,然后转化烟草(Nicotianatabacum)叶片获得再生植株。经PCR、PCR-Southern、Northern杂交和蛋白质检测,证明patatin classⅠ基因cDNA已整合到烟草基因组中并在转基因植株中转录和表达。酯酰水解酶(lipidacylhydrolase,LAH)活性分析显示,转基因烟草植株的叶片中LAH活性明显提高。Patatin is the trivial name for a family of glycoproteins that accounts for up to 40% of the soluble protein in potato (Solanum tuberosum L.) tubers. Unlike most other storage proteins, patatin has a lipid acylhydrolase (LAH) activity. It is proposed that patatin may be involved in particular biological processes, especially tuber formation. Despite a detailed molecular and biochemical analysis of patatin, its physiological role and function, especially in potato tuber formation and regulation have not been elucidated. In order to approach this question, we have isolated a classⅠpatatin cDNA (GenBank accession No. AF498099) from a cDNA library constructed from wild diploid potato species Solanum chaconse under tuber inducing conditions. We constructed the expression vector pBSSP by fusing the patatin class I cDNA with CaMV 35S promoter and introduced it into tobacco without patatin gene to prove its expression and LAH activity. The expression vector pBSSP was introduced into Agrobacterium tumefaciens strain LBA4404 by electroporation and then transferred into tobacco (Nicotiana tabacum ) via Agrobacterium tumefaciens system. Leaves from 4-week-old plant grown in vitro of tobacco line T1 were used for transformation. Transformants were selected on MS medium containing 2.25 mg/L BA, 0.3 mg/L NAA, 100 mg/L kanamycin and 400 mg/L carbenicillin. When green shoots reached 1~1.5 cm, transferred them to the selective rooting MS medium supplemented with 50 mg/L kanamycin and 200 mg/L carbenicillin. A total of 45 kanamycin resistance plants were obtained. In order to prove patatin class Ⅰ cDNA was integrated into genome of tobacco, DNA was isolated from putative transformed and control tobacco plants. PCR analysis using patatin class Ⅰ cDNA specific primers was performed on 45 putatively transformed plants. There were 40 transformed plants showed a 500 bp amplification product which was missing in non-transformed control plants. The PCR amplification results were conformed by PCR-Southern blot analysis. The PCR prod

关 键 词：烟草 马铃薯块茎特异蛋白 patatinclassI基因 酯酰水解酶 

分 类 号：S572[农业科学—烟草工业] Q943.2[农业科学—作物学]