烟草C3HC4型锌指蛋白的原核表达及转基因植株功能研究

生物技术通报 ›› 2014, Vol. 0 ›› Issue (7): 100-105.

烟草C3HC4型锌指蛋白的原核表达及转基因植株功能研究

伍文宪^{1, 2}, 杨秀芬¹, 刘勇², 邱德文¹

1. 中国农业科学院植物保护研究所, 北京 100081；
2. 四川省农业科学院植物保护研究所, 成都 610066

收稿日期:2014-02-11 出版日期:2014-07-15 发布日期:2014-07-16
作者简介:伍文宪, 硕士研究生, 研究方向：微生物分子生物学与基因工程；E-mail：wuwenxian07640134@163.com
基金资助:
国家自然科学基金项目（31371984）

Prokaryotic Expressing and Functional Analysis of C3HC4-type Zinc Finger Protein in Nicotiana benthamiana

Wu Wenxian^{1, 2}, Yang Xiufen¹, Liu Yong², Qiu Dewen¹

1. Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100081；
2. Institute of Plant Protection, Sichuan Academy of Agricutural Sciences, Chengdu 610066

Received:2014-02-11 Published:2014-07-15 Online:2014-07-16

摘要/Abstract

摘要： 植物中C3HC4型RING锌指蛋白在泛素化、光形态建成、抗逆及生长发育等过程中均起到非常重要的作用。首次从本生烟中扩增获得一个锌指蛋白基因的完整阅读框, 编码203个氨基酸, 含有C3HC4型锌指结构域, 命名为NbZFP1。将NbZFP1基因分别克隆到原核表达系统载体pGEX-6P-2以及pMAL-C2X中, 成功构建了重组表达载体pGEX-6P-2-NbZFP1与pMAL-C2X-NbZFP1, 经IPTG获得了大量可溶性表达的NbZFP1融合蛋白。将NbZFP1基因克隆于植物表达载体pBI121中, 通过农杆菌介导法获得转基因烟草, PCR检测及Southern blot分析表明NbZFP1基因已整合到烟草基因组中, 转NbZFP1基因烟草表现出株型紧凑, 节间缩短, 茎干粗壮和对TMV抗性增强。

关键词: 锌指蛋白, 重组表达, 转基因烟草, 抗病性

Abstract: C3HC4-type RING finger genes comprise a large family in the plant kingdom and play important roles in various physiologic processes of plant life. In this study, a zinc finger gene was isolated from Nicotiana benthamiana. Sequence analysis indicated that the gene encodes a 24 kD protein with 203 amino acids and contains one typical C3HC4 zinc finger domain and named NbZFP1. The entire coding region of NbZFP1 gene was then cloned into the bacterial expression vectors pGEX-6P-2 and pMAL-C2X. After IPTG induction, the recombinant protein was analyzed by SDS-PAGE, which showed that the expected protein was expressed at a high level in bacterial cells. NbZFP1 gene was cloned into the over-expression vector pBI121, and then transfered into Nicotiana benthamiana through Agrobacterium tumefacien-mediated transformation. According to PCR and Southern blot detection, NbZFP1 gene was successfully integrated into Nicotiana benthamiana genome. Regeneration plants showed type compactness, internode shorten and stem sturdy, also enhanced disease resistance against tobacco mosaic virus（TMV）.

Key words: Zinc finger protein, Recombinant expression, Transgenic tobacco, Disease resistance

伍文宪, 杨秀芬, 刘勇, 邱德文. 烟草C3HC4型锌指蛋白的原核表达及转基因植株功能研究[J]. 生物技术通报, 2014, 0(7): 100-105.

Wu Wenxian, Yang Xiufen, Liu Yong, Qiu Dewen. Prokaryotic Expressing and Functional Analysis of C3HC4-type Zinc Finger Protein in Nicotiana benthamiana[J]. Biotechnology Bulletin, 2014, 0(7): 100-105.

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