Construction of Double Cross-over Expression Vector for Chloroplast Multicistron in Brassica napus L.

作     者：武玉永 姚庆收 马立新 Yuyong WU;Qingshou YAO;Lixin MA

作者机构：滨州医学院医学遗传学教研室山东烟台264003 湖北大学生命科学学院分子微生物学与基因工程实验室湖北武汉430062 

基　　金：Supported by National 863 Project of China (2002AA227011) Natural Science Foundation of Hubei Province (2003ABAI18) Natural Science Foundation of Shandong Province (ZR2010HQ054) 

出 版 物：《Agricultural Science & Technology》 (农业科学与技术（英文版）)

年 卷 期：2013年第14卷第3期

页      码：402-406页

摘      要：[Objective] This study aimed to construct Brassica napus chloroplast multi- cistron double cross-over expression vector, to lay the foundation for the genetic engi- neering research of Brassica napus chloroplast. [Method] Two primers were designed based on the known Brassica napus chloroplast DNA sequences AF267640 and Z50868 in GenBank. By using PCR method, two Brassica napus L. chloroplast DNA fragments were obtained, which were named RbcL and ACCD. The two Brassica na- pus chloroplast DNA homologous fragments were then cloned into plasmid pMD18-T to obtain recombinant plasmid pHBM715. Tandem expression cassette harboring spectinomycin-resistant gene aadA, mannanase gene man and green fluorescent pro- tein gene gfp was cloned into the plasmid pHBM715, thereby constructing Brassica napus chloroplast multicistron double cross-over expression vector pHBM716, which was transformed into Escherichia coil for expression and identification. [Result] Plate qualitative analysis was conducted for the functional identification of expression cas- sette in the constructed Brassica napus chloroplast multicistron double cross-over ex- pression vector, results showed that the three genes of the same multicistron were all expressed in E. coil [Conclusion] This study successfully constructed Brassica napus chloroplast multicistron double cross-over expression vector, which laid the foundation for the genetic engineering of Brassica napus chloroplast.

主 题 词：Brassica napus L. Chloroplast DNA Multicistron Double cross-over Expression vector Functional identification 

学科分类：09[农学] 0901[农学-植物生产类] 

D　O　I：10.3969/j.issn.1009-4229(e).2013.03.004

馆 藏 号：203627283...