葡萄硫氧还蛋白基因(VvTrx)的克隆、表达和原核表达

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摘要硫氧还蛋白(thioredoxin, Trx)是一类高度保守的低分子量蛋白，广泛存在于植物、动物、细菌、酵母中，具有维持生物体内氧化还原平衡和调控生物信号传导等多种功能。本研究克隆了葡萄(Vitis vinifera) VvTrx 基因989 bp的gDNA 序列(GenBank 登录号: EU280166)和561 bp的cDNA序列 (GenBank 登录号: EU280164)，开放阅读框(open reading frame, ORF)为381 bp，编码126个氨基酸。氨基酸多序列比对和进化树分析发现，无核白(Vitis vinifera cv. Thompson Seedless)VvTrx基因的活性功能位点为WCIPS，属于h型硫氧还蛋白的第四亚族。实时荧光定量PCR(quantitative Real-time PCR, qRT-PCR)分析表明，在无核白和黑比诺(Vitis vinifera cv. Pinot Noir)的胚珠发育不同时期中， VvTrx基因在花后40 d表达量均达到整个胚珠发育期的最高水平，二者差异极其显著 (P＜0.001)，且无核白的表达量约为黑比诺的5倍。将VvTrx克隆到原核表达载体pET-32a(+)构建pET-32a(+)/VvTrx并转入大肠杆菌(Escherichia coli)BL21中，经异丙基硫代半乳糖苷(isopropyl β-D-1-thiogalactopyranoside, IPTG)诱导后，获得30 kD的可溶性融合蛋白。研究表明，VvTrx基因与无核葡萄的胚败育存在一定关系，研究结果为进一步揭示VvTrx基因在无核葡萄胚珠败育过程中的分子机理和无核葡萄育种提供研究方向。

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关键词 ：葡萄, 硫氧还蛋白(Trx), 克隆, 表达分析, 原核表达

Abstract：Thioredoxin (Trx) is a kind of highly conservative and low molecular weight proteins, which widely exists in plants, animals, bacteria and yeasts. It maintains the redox equilibrium of organisms, which regulates and controls biological signal transduction and other functions. This study cloned VvTrx gene from grape (Vitis vinifera) gDNA sequence (GenBank No. EU280166) of 989 bp and cDNA sequence (GenBank No. EU280164) of 561 bp and the open reading frame (ORF) was 381 bp, encoding 126 amino acids. The multiple sequence alignment and phyogenetix tree showed that the active site of VvTrx gene in Thompson Seedless (Vitis vinifera cv.) was WCIPS and belonged to the subgroup Ⅳ in Trx-h. Analysis of quantitative Real-time PCR(qRT-PCR) showed that, in different periods of the ovule development of both Thompson Seedless and Pinot Noir, the expression of VvTrx gene reached the highest level in 40 d after flowering. Besides, the difference between them was extremely significant (P＜0.001) and the expression of Thompson Seedless was about 5 times of Pinot Noir. After that, the VvTrx gene was cloned into the prokaryotic expression vector of pET-32a(+) to construct pET-32a(+)/VvTrx, and a 30 kD solubility fusion protein expressed in the Escherichia coli BL21 through isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. The study showed that the VvTrx gene had a certain relation to the process of seedless grape ovules abortion, which provides the research directions to the molecular mechanism of VvTrx gene in seedless grape embryo abortion process and the breeding of seedless grape.

Key words： Grape Thioredoxin(Trx) Cloning Expression analysis Fusion expression

收稿日期: 2015-02-16 出版日期: 2015-07-09

基金资助:国家自然科学基金项目<葡萄胚珠特异表达基因VvABCG2跨膜域变异对种子发育的影响>;国家现代农业产业技术体系建设专项<无核葡萄育种>;陕西省农业攻关项目<葡萄丰产栽培及配套技术研究与示范>

通讯作者: 张朝红 E-mail: zhangchaohong@nwsuaf.edu.cn

引用本文:

张小莹唐玉瑾王刚王跃进张朝红. 葡萄硫氧还蛋白基因(VvTrx)的克隆、表达和原核表达[J]. , 2015, 23(9): 1131-1140.

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http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2015/V23/I9/1131

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