菊苣叶绿体同源片段的克隆及多顺反子定点整合表达载体的构建

doi:10.7668/hbnxb.2010.01.003

华北农学报 ›› 2010, Vol. 25 ›› Issue (1): 11-17. doi: 10.7668/hbnxb.2010.01.003

菊苣叶绿体同源片段的克隆及多顺反子定点整合表达载体的构建

王玉华¹, 韩晓玲³, 黄丛林², 贾敬芬¹

1. 西北大学,生命科学学院,陕西省生物技术重点实验室,西部资源生物与现代生物技术教育部重点实验室, 陕西西安 710069;
2. 西安力邦制药有限公司,医药科学研究所, 陕西西安 710075;
3. 北京市农林科学院,北京农业生物技术研究中心, 北京 100097

收稿日期:2009-12-16 出版日期:2010-02-28
通讯作者: 贾敬芬(1938-), 女,河北深泽人,教授,博导,主要从事植物细胞工程研究.
作者简介:王玉华(1975-),女,山东肥城人,讲师,博士,主要从事植物基因工程研究.
基金资助:
国家自然科学基金(30900914);陕西省教育厅专项(09JK777);西北大学西部资源生物与现代生物技术教育部重点实验室开放基金.

Cloning of Homologous Targeting Sequences of Chicory and Construction of Multicistron Chloroplast Site-Specific Integration Expression Vector

WANG Yu-hua1¹, HAN Xiao-ling3³, HUANG Cong-lin2², JIA Jing-fen1¹

1. College of Life Sciences, Northwest University, Shaanxi Provincial Key Laboratory of Biotechnology, Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, Xi′an 710069, China;
2. Beijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China;
3. Pharmaceutical Sciences Research Institute, Xi′an Libang Pharmaceutical Co., Ltd., Xi′an 710075, China

Received:2009-12-16 Published:2010-02-28

摘要/Abstract

摘要： 利用叶绿体基因组进化中高度保守的特点,根据烟草叶绿体基因组全序列设计合成引物,从菊苣叶绿体基因组中扩增包括rps-rps12-trnV-16S-rDNA基因在内的一段序列(GenBank登录号为GQ19948),并以此作为定点整合外源基因的同源重组片段.以叶绿体基因强启动子prrn驱动选择标记基因aadA及报告基因gfp构建多顺反子表达盒prrn-aadA-gfp-psbA-3′,然后将表达盒克隆进菊苣叶绿体同源片段中,获得菊苣叶绿体定点整合表达载体pJAG,酶切分析证明所构建的载体符合预期设计.该载体的构建为建立菊苣的叶绿体转化体系奠定基础,为进一步通过叶绿体基因工程手段将更多感兴趣的基因导入菊苣进行遗传改良,或以菊苣叶绿体作生物反应器生产动物口服疫苗等搭建技术平台.

关键词: 菊苣, 叶绿体同源片段, 多顺反子, 定点整合表达载体

Abstract: Since chloroplast genomes were highly conservative in evolution,PCR primers were designed basing on the corresponding sequences in the tobacco chloroplast genome.The rps7-rps12-trnV-16S rDNA targeting region(GenBank Accesion Number was GQ199478) was cloned from chicory chloroplast genome,which was used as homologous targeting sequences,and the site-specific integration chicory-specific chloroplast expression vector named pJAG that carries the multicistron expression cassette of prrn-gfp-aadA-psbA-3′ was constructed.The results of restriction analysis were in accord with the desired.This chloroplast expression vector was desirable to be applied in both establishment of chicory chloroplast transformation system and traits improvement of chicory or using chicory chloroplast as bioreactor to produce edible vaccine for animals via plastid genetic transformation.

Key words: Chicory, Chloroplast homologous fragments, Multicistron, Site-specific integration expression vector

中图分类号:

Q789

王玉华, 韩晓玲, 黄丛林, 贾敬芬. 菊苣叶绿体同源片段的克隆及多顺反子定点整合表达载体的构建[J]. 华北农学报, 2010, 25(1): 11-17. doi: 10.7668/hbnxb.2010.01.003.

WANG Yu-hua1,HAN Xiao-ling3,HUANG Cong-lin2,JIA Jing-fen1. Cloning of Homologous Targeting Sequences of Chicory and Construction of Multicistron Chloroplast Site-Specific Integration Expression Vector[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2010, 25(1): 11-17. doi: 10.7668/hbnxb.2010.01.003.

http://www.hbnxb.net/CN/Y2010/V25/I1/11

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