玉米叶绿体表达载体构建及绿色荧光蛋白基因瞬时表达检测

doi:10.7668/hbnxb.2008.04.017

华北农学报 ›› 2008, Vol. 23 ›› Issue (4): 72-76. doi: 10.7668/hbnxb.2008.04.017

所属专题： 玉米；生物技术；

玉米叶绿体表达载体构建及绿色荧光蛋白基因瞬时表达检测

赵彤^1,2, 黄丛林¹, 张秀海¹, 于荣², 王永勤¹, 吴忠义¹

1. 北京市农林科学院北京农业生物技术研究中心,北京 100097;
2. 首都师范大学生命科学学院,北京 100037

收稿日期:2008-03-31 出版日期:2008-08-28
通讯作者: 吴忠义(1969-),男,福建德化人,副研究员,主要从事植物分子生物学的研究
作者简介:赵彤(1982-),女,北京人,在读硕士,主要从事植物分子生物学的研究
基金资助:
北京市自然科学基金项目(5062012);国家自然科学基金项目(30400228,30600318)

Construction of Maize Chloroplast Expression Vector and Transient Expression in Maize Chloroplasts by Detecting GFP

ZHAO Tong^1,2, HUANG Cong-lin¹, ZHANG Xiu-hai¹, YU Rong², WANG Yong-qin¹, WU Zhong-yi¹

1. Beijing Research Center of Agro-Biotechnology,Beijing Academy of Agriculture and Forestry Sciences,Beijing 100097,China;
2. College of Life Science,Capital Normal University,Beijing 100037,China

Received:2008-03-31 Published:2008-08-28

摘要/Abstract

摘要： Tps1是生物保护物质海藻糖的关键合酶基因。构建含Tps1的玉米叶绿体表达载体,首先从玉米叶绿体基因组中克隆16S/trnI-trnA/23S片段,作为定点整合同源片段;然后将编码酵母海藻糖合酶基因Tps1表达盒(Prrn-Tps1-TpsbA-ter)、草丁膦抗性基因Bar表达盒(RpsbApro-Bar-RpsbAter)、卡那霉素抗性基因Npt II和绿色荧光蛋白基因Gfp构建的融和基因表达盒(Prrn-Npt II/Gfp-RrbcLter)一起克隆到定点整合同源片段之间,构建了玉米叶绿体的稳定表达载体mTKGB。通过基因枪轰击法将mTKGB转化到玉米幼嫩叶片中,培养2 d后,在激光扫描共聚焦显微镜下观测到玉米一些细胞的叶绿体中有很强的GFP绿色荧光,结果表明构建的载体可在玉米叶绿体中表达。

关键词: 玉米, 叶绿体表达载体, 瞬时表达, GFP荧光

Abstract: Tps1 encodes a key enzyme in trehalose synthesis1Maize chloroplast expression vector harboring the yeast Tps1 is constructed in this paper1Firstly, the homologous fragment 16S/ trnI2trnA/ 23S was amplified frommaize chloro2 plast genome by PCR for site2specific integration. Secondly, the yeast Tps1 cassette ( Prrn2 Tps12 Tpsb A2ter ), phos2 phinothricin2resistant gene Bar cassette( Rpsb A pro2 Bar2Rpsb Ater) and Npt / G fp cassette consistingof kanamycin2resistant gene Npt and green fluorescent protein gene G fp ( Prrn2 Npt/ G fp2 RrbcLter) were constructed respectively1 Finally, the three expression cassettes were cloned into the homologous fragment to obtain chloroplast stable expression vector mTKGB.Then mTKGBwas introduced into young leaves of maize through particle bombardment1Two days later, strong green fluorescence was observed in the chloroplasts of some bombarded leaves under confocal laser scanning microscope, indicating that m TKGB could be expressed in maize chloroplasts1The results of this paper provide a strong foundation for genetic engineering drought2resistant maize through chloroplast transformation in the future.

Key words: Maize, Chloroplast expression vector, Transient expression, GFP

中图分类号:

Q786

赵彤, 黄丛林, 张秀海, 于荣, 王永勤, 吴忠义. 玉米叶绿体表达载体构建及绿色荧光蛋白基因瞬时表达检测[J]. 华北农学报, 2008, 23(4): 72-76. doi: 10.7668/hbnxb.2008.04.017.

ZHAO Tong, HUANG Cong-lin, ZHANG Xiu-hai, YU Rong, WANG Yong-qin, WU Zhong-yi. Construction of Maize Chloroplast Expression Vector and Transient Expression in Maize Chloroplasts by Detecting GFP[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2008, 23(4): 72-76. doi: 10.7668/hbnxb.2008.04.017.

http://www.hbnxb.net/CN/Y2008/V23/I4/72

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玉米叶绿体表达载体构建及绿色荧光蛋白基因瞬时表达检测

Construction of Maize Chloroplast Expression Vector and Transient Expression in Maize Chloroplasts by Detecting GFP

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玉米叶绿体表达载体构建及绿色荧光蛋白基因瞬时表达检测

Construction of Maize Chloroplast Expression Vector and Transient Expression in Maize Chloroplasts by Detecting GFP

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