摘要
[目的]为构建乙酰辅酶A羧化酶的叶绿体表达载体奠定基础：[方法]以从甘蓝型油菜叶片中提取的叶绿体基因组DNA为模板进行PCR扩增，克隆羧基转移酶β亚基基因.以从开花20～29d后油菜幼胚中提取的总RNA为模板进行RT-PCR扩增，克隆生物素羧化酶和生物素羧基载体蛋白的。cDNA序列。最后，乙酰辅酶A羧化酶的3个亚基基因被克隆到大肠杆菌表达载体中：[结果]SDS-PAGE分析结果表明乙酰辅酶A羧化酶的3个亚基基因分别被克隆到大肠杆菌表达载体中。乙酰辅酶A羧化酶的3个亚基基因在大肠杆菌中的表达产物分别为76．6、44．0和81．5kD，其融合蛋白分别占总菌体蛋白的12％、10％和6％。
[ Obiective] The research aimed to lay the foundation for constructing the chloroplast expression vector of aeetyl-CoA carboxylase, [Method] The chlomplast genomic DNA extracted from the leaves of Brassica napus L. was taken as template for PCR amplification and the β -subunit gene of carboxyltransferase was cloned. Total RNA extracted from immature embryo of rape after flowering for 20 - 29 days was taken as template for RT-PCR amplification trod the cDNA sequences of biotin carboxylase and biotin carboxyl carrier protein were cloned. Finally, 3 subunit genes of acetyl-CoA carbaxylase were cloned into the expression vector of E. eoli. [ Result] The results of SDS-PAGE analysis showed that 3 subunit genes of acetyl-CoA carboxylase were successfully cloned into the expression vector of E. coli. The expression products of 3 subunit genes of acetyl-CoA carboxylase in E. coli were 76.6, 44.0 and 81.5 kD resp. and their fusion proteins were 12%, 10% and 6% of total bacterial protein resp. [ Conclusiou] The eDNA sequence of biotin carboxyl cartier proteiu had greater difference among different varieties of B. napus L.
出处
《安徽农业科学》
CAS
北大核心
2008年第10期4002-4006,共5页
Journal of Anhui Agricultural Sciences
基金
湖北省自然科学基金项目(2003ABA118)
