Bt-<em>Cry5Aa</em>杀虫基因的原核表达、纯化及其在棉花中的鉴定

doi:10.3969/j.issn.1674-7968.2020.09.005

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摘要单一转苏云金芽胞杆菌(Bacillus thuringiensis, Bt)基因Cry1Ab/Cry1Ac抗虫棉(Gossypium)作为我国生产上大面积种植作物,随着种植时间及面积的增加导致害虫对Bt毒蛋白的抗性增强迫使新型Bt基因研究工作成为解决问题的关键。Cry5Aa是一种具有兼抗鳞翅目和线虫的新型Bt杀虫基因,由湖南农业大学棉花研究所成功转入棉花,并获得稳定遗传的'JX0010'、'JX0020'品系,本研究构建了Cry5Aa全长基因原核表达载体,优化了原核表达诱导条件,并且对其在转Bt-Cry5Aa基因棉花品系中的表达进行了鉴定。结果表明,在37 ℃ 220 r/min 1.0 mmol/L异丙基-β-D-硫代半乳糖苷(isopropyl-beta-D-thiogalactopyranoside, IPTG)条件下诱导4 h,其蛋白表达量最高。通过Western blot分析,发现蛋白分子量为79 kD,与理论值一致;同时,该蛋白以包涵体形式存在,通过谷胱甘肽-Sepharose4B小颗粒亲和层析纯化,蛋白纯度可达95%以上;转Bt-Cry5Aa基因棉花品系的鉴定实验表明：Cry5Aa基因能在转基因品系'JX0010'、'JX0020'中表达杀虫毒蛋白,且'JX0010'的Cry5Aa毒蛋白表达量在棉花不同时期及不同部位均高于'JX0020',且在生长期以蕾期为最高表达,植株部位上以叶片为最高表达。本研究构建了Bt-Cry5Aa杀虫基因的原核表达载体,获得了Cry5Aa的纯化蛋白,初步对其在棉花中的表达进行了鉴定,为对Bt-Cry5Aa杀虫基因的抗虫性、安全性等转基因检测及进一步在棉花中的应用提供了理论基础。

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	马肖
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	周仲华

关键词 ：苏云金芽胞杆菌(Bt), Cry5Aa基因, 原核表达, 蛋白纯化, 表达鉴定

Abstract：The single transgenic Bacillus thuringiensis (Bt) gene Cry1Ab/Cry1Ac insect-resistant cotton (Gossypium) is a large-scale planted crop in China. With the increase of planting time and area, the resistance of pests to Bt toxin protein increases, new Bt gene research work is necessary. Cry5Aa is a novel Bt insecticidal gene with resistance to both Lepidoptera and Nematodes, was successfully transferred into cotton by Cotton Research Institute of Hunan Agricultural University and a stable genetic 'JX0010', 'JX0020' strain was obtained. In this study, the Cry5Aa full-length gene prokaryotic expression vector was constructed, the prokaryotic expression induction conditions were optimized, and its expression in the Bt-Cry5Aa gene of transgenic cotton line was identified. The results showed that the protein expression was the highest when induced at 37 ℃ with 220 r/min 1.0 mmol/L isopropyl-beta-D-thiogalactopyranoside (IPTG) for 4 h. Through Western blot experiment, it was found that the molecular weight of the protein was 79 kD, which was consistent with the theoretical value; at the same time, the protein existed in the form of inclusion body and was purified by Glutathione-Sepharose 4B small particle affinity chromatography. The identification experiment of transgenic cotton lines with Bt-Cry5Aa gene showed that Cry5Aa gene could express insecticidal protein in transgenic line 'JX0010', 'JX0020', and the expression of Cry5Aa toxin protein of 'JX0010' was higher than that of 'JX0020' in different periods and different parts of cotton. The expression in bud stage was the highest, and the spatial distribution was the highest in leaves. In this study, the prokaryotic expression vector of Bt-Cry5Aa insecticidal gene was constructed, the purified protein of Cry5Aa was obtained, and its expression in cotton was preliminarily identified, which provides a theoretical basis for the further application of Bt-Cry5Aa insecticidal gene in cotton.

Key words： Bacillus thuringiensis (Bt) Cry5Aa gene Prokaryotic expression Protein purification Expression identification

收稿日期: 2020-01-08

ZTFLH:	S562
	Q789

通讯作者: **,zhanqiuping@hunau.edu.cn;zhouzhonghua1976@hotmail.com.
*同等贡献作者

引用本文:

马肖, 赵世浩, 王峰, 贺文, 陈金湘, 张秋平, 周仲华. Bt-Cry5Aa杀虫基因的原核表达、纯化及其在棉花中的鉴定[J]. 农业生物技术学报, 2020, 28(9): 1567-1575.
MA Xiao, ZHAO Shi-Hao, WANG Feng, HE Wen, CHEN Jin-Xiang, ZHANG Qiu-Ping, ZHOU Zhong-Hua. Prokaryotic Expression and Purification of Bt-Cry5Aa Insecticidal Gene and Its Identification in Gossypium. 农业生物技术学报, 2020, 28(9): 1567-1575.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2020.09.005 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2020/V28/I9/1567

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