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.
出处
《热带作物学报》
CSCD
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
