Δ~6-脂肪酸脱氢酶基因克隆及其共转化表达载体的构建  

Cloning of Δ~6-Desaturase Gene and Construction of Its Co-transformation Expression Vector

作　　者：张秀春[1] 林俊芳[1,2] 郭丽琼[1,2] 

机构地区：[1]中国热带农业科学院热带作物生物技术国家重点实验室 [2]华南农业大学食品学院生物工程系广东广州510640

出　　处：《热带作物学报》2004年第4期63-67,共5页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金(30371000);广东省自然科学基金(032239);华南农业大学校长基金(5100-k03003)资助项目

摘　　要：采用RT-PCR的方法从海南玻璃苣中克隆了Δ6-脂肪酸脱氢酶基因(Δ6-dsa)。DNA序列测定结果表明,Δ6-dsa全长1347bp编码448个氨基酸。Δ6-dsa序列在genbank中采用BLAST程序进行同源序列分析,结果表明,Δ6-dsa核苷酸序列与克隆自美国德克萨斯州的玻璃苣Δ6-脂肪酸脱氢酶基因的核苷酸序列完全相同。Δ6-dsa克隆进T-easyVector后形成质粒pGΔ6-DSA。把质粒pGΔ6-DSA上的Δ6-脂肪酸脱氢酶基因片段克隆进含有T-DNA和35S启动子的质粒pGIN22中,形成含有目的基因的表达载体pGIN23。把表达载体pGIN23与另一个含有T-DNA、启动子、筛选标记基因的表达载体pLPN28进行亚克隆,使得筛选标记基因和Δ6-dsa分别插入到同一质粒的2个相互独立的T-DNA内,构建成共转化表达载体pLIN61。RT-PCR was used for the amplification of Δ6-desaturase gene(Δ6-dsa )from Borage in Hainan, China.DNA sequencing results showed that Δ6-desaturase gene was composed of 1 344 base pairs and encoded 448 amino acid residues. Sequence alignments with BLAST revealed that the nucleotide sequence and its deduced amino acid sequence of Δ6-desaturase gene from Chinese Borage were completely homologic to those from American Borage.Δ6-dsa was cloned into T-easy vector as pGΔ6-DSA.The fragment containing Δ6-dsa on pGΔ6-DSA was sub-cloned into pGIN22 containing T-DNA and 35S promoter as expression vector pGIN23.And pGIN23 was further sub-cloned with another expression vector pLPN28 containing another T-DNA,promoter and selectable marker gene.Then a co-transformation expression vector, designated as pLIN61,was constructed with two separate T-DNAs and expression cassettes,on which one contained Δ6-desaturase gene and another contained selectable marker gene.

关 键 词：△^6-脂肪酸脱氢酶 表达载体 克隆 共转化 筛选标记基因 酶基因 DNA DSA 质粒 玻璃苣 

分 类 号：S565.1[农业科学—作物学] Q78[生物学—分子生物学]