桑树1,5-二磷酸核酮糖羧化酶活化酶基因cDNA片段的克隆及原核表达与植物反义表达载体的构建  

Cloning and Prokaryotic Expression of Mulberry Rubisco Activase cDNA Fragment and Construction of Antisense Expression Vector

作　　者：冀宪领[1] 盖英萍[2] 王洪利[1] 牟志美[1] 

机构地区：[1]山东农业大学林学院蚕学系,山东泰安271018 [2]山东农业大学作物生物学国家重点实验室,山东泰安271018

出　　处：《蚕业科学》2009年第1期6-12,共7页ACTA SERICOLOGICA SINICA

基　　金：山东省农业良种产业化开发项目[编号(2008)167]

摘　　要：1,5-二磷酸核酮糖羧化酶活化酶(RCA)对桑树的光合作用具有重要调节作用。以桑树幼叶为材料分离mRNA,反转录合成cDNA,以cDNA第1链为模板,根据RCA的保守区域设计1对兼并引物,经PCR扩增获得RCA的基因功能区中间片段。对得到的桑树RCA的cDNA片段编码氨基酸进行BLAST分析表明,其与GenBank中其它植物来源的RCA有较高同源性。将RCA的部分编码区插入原核表达载体pET30a(+),并转化到大肠杆菌菌株BL21中,经IPTG诱导,RCA的部分编码区在BL21菌株成功表达。将得到的RCA基因片段反向插入植物表达载体,构建了RCA基因反义表达载体pBI121-RCA,以利于进一步阐明RCA与1,5-二磷酸核酮糖羧化酶相互作用和调控关系以及用基因工程手段深入探讨桑树光合作用机制。mRNA was isolated from young mulberry leaves and reverse-transcribed to yield the first strand cDNA. Degenerate primers were designed based on conserved regions among the known RCA （ribulose-1,5-bisphosphate carboxylase activating enzyme） sequences and were used to amplify RCA fragment by PCR using the first strand cDNA as template. Amino acid sequence analysis indicated that the sequence deduced from the cloned cDNA fragment showed high similarity with other plant RCAs. After the RCA coding region was inserted into an expression vector [ pET30a（ ＋ ） ] and transformed into Escherichia coli BL21, the encoded protein was successfully expressed after induction with IPTG. Besides, an antisense expression vector with the same fragment under control of 35S promoter was constructed. The present study provides useful information for understanding the interaction between RCA and Rubisco and for further investigation on photosynthetic mechanism using genetic engineering methods.

关 键 词：桑树 1 5-二磷酸核酮糖羧化酶活化酶 基因克隆 原核表达 反义表达载体 

分 类 号：S888.2[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]