银杏HDR基因的克隆与功能分析  

Cloning and Functional Analysis of HDR Gene from Ginkgo biloba L.

作　　者：杨颖舫[1] 杨春贤[1] 冯国庆[1] 成瑜[1] 李郑娜[1] 陈敏[2] 廖志华[1] 

机构地区：[1]西南大学生命科学学院,重庆400715 [2]西南大学药学院,重庆400715

出　　处：《安徽农业科学》2010年第13期6663-6665,6705,共4页Journal of Anhui Agricultural Sciences

基　　金：国家自然科学基金(30500303)

摘　　要：[目的]获得银杏HDR基因的克隆并研究HDR基因的功能。[方法]采用RT-PCR技术获得银杏HDR的全长cDNA,命名为Gb-HDR(GenBank登录号:DQ364231),该基因cDNA全长为1827bp,包含1425bp的开放阅读框,编码474个氨基酸残基的蛋白;并构建GbHDR的原核表达载体pTrcGbHDR,与原核表达载体pAC-BETA共转入大肠杆菌XL1-Blue,获得β-胡萝卜素工程菌。[结果]克隆获得实际长度为1441bp的GbHDR基因,该序列包含1425bp的HDR基因ORF,编码474个氨基酸残基的蛋白,其预测分子量为53.2kD,等电点预测为5.76。功能互补分析表明,GbHDR能推动工程菌XL1-Blue+pTrcGbHDR+pAC-BETA超量表达β-胡萝卜素,在颜色互补平板上呈现β-胡萝卜素特有的橘黄色,证实GbHDR具有典型的HDR基因功能。[结论]获得1株可高量积累β-胡萝卜素的大肠杆菌工程菌,为最终实现β-胡萝卜素代谢工程提供侯选基因和作用靶点。[ Objective ] The aim was to obtain cloning of HDR gene from Ginkgo biloba L. and study its function. [ Method] The coding sequence of HDR gene was cloned from Ginkgo biloba L. by reverse transcription polymerase chain reaction, which was designated as GbHDR （ GenBank accession No. ： DQ364231 ）. The cDNA full-length of GbHDR is 1 827 bp containing a 1 425-bp open reading frame （ORF） enco- ding a 474-amino-acid polypeptide and constructed into the prokaryotic expression vector pTrcGbHDR. The 13-carotene biosynthetic pathway in E. coli strain XL1-Blue was reconstructed by transforming with pAC-BETA . This engineered XL1-Blue was transformed with pTrcGbHDR. [ Result] A 1 441 bp GbHDR was obtained containing a 1 425-bp ORF encoding a 474-amino-acid residues of the protein, the predicted molecular weight was 53.2 kD, and predicted isoelectric point was 5.76. Functional complementation assay indicated that GbHDR could promote the β-carotene accumulation in engineered XL1-Blue haboring pTrcGbHDR and pAC-BETA, and as a result the engineered bacteria showed the brightly orange given by β-carotene. This suggested that GbHDR had the typical function of known HDR genes. [ Conclusion] A engineered bacteria of E. coli which could highly accumulate β-carotene was obtained, which will provide candidate genes and targets for realizing β-caro- tene metabolic engineering.

关 键 词：HDR基因 基因克隆 β-胡萝卜 功能互补 银杏 

分 类 号：Q943.2[生物学—植物学]