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柑橘大实蝇GSTd1基因的克隆、原核表达及重组蛋白GSTd1的酶学特征

引用本文：刘毅,田晓丽,毛永娜,桂连友,王福莲,张国辉.柑橘大实蝇GSTd1基因的克隆、原核表达及重组蛋白GSTd1的酶学特征.植物保护学报,2023,50(5):1269-1279

DOI：10.13802/j.cnki.zwbhxb.2023.2022093

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作者	单位	E-mail
刘毅	长江大学农学院, 长江大学昆虫研究所, 湖北荆州 434025
田晓丽	长江大学生命科学学院, 湖北荆州 434025
毛永娜	湖北省林业科学研究院荆州分院, 荆州 434020
桂连友	长江大学农学院, 长江大学昆虫研究所, 湖北荆州 434025
王福莲	长江大学农学院, 长江大学昆虫研究所, 湖北荆州 434025
张国辉	长江大学农学院, 长江大学昆虫研究所, 湖北荆州 434025	zhangguohuiji@163.com

中文摘要:为深入研究柑橘大实蝇Bactrocera minax嗅觉系统中气味信号降解机制，对柑橘大实蝇头部转录组进行分析，筛选谷胱甘肽S-转移酶（glutathione S-transferase，GST）基因，对其进行序列测定和分子生物学分析，利用实时荧光定量PCR技术检测该基因在柑橘大实蝇成虫各组织中的表达模式，并对该基因进行克隆和原核表达，采用Western blot对获得的编码蛋白GSTd1进行检测，并分析该蛋白的酶学特征。结果显示，成功获得1条具有完整开放阅读框的GST基因，将其命名为GSTd1，该基因完整开放阅读框为624 bp，编码207个氨基酸残基，预测蛋白分子量为23.72 kD，等电点为6.16，无信号肽，不含跨膜结构域，属于胞质型GST蛋白。序列比对和系统进化树分析结果表明柑橘大实蝇GSTd1属于昆虫特有的GST蛋白的Delta亚家族成员。柑橘大实蝇GSTd1基因在柑橘大实蝇成虫触角中的相对表达量最高。柑橘大实蝇GSTd1重组蛋白具有催化其通用底物1-氯-2，4-二硝基苯（1-chloro-2,4-dinitrobenzene，CDNB）的能力，最大反应速度和米氏常数分别为11.76 μmol·mg^-1·min^-1和0.224 mmol/L，当pH为7.5，温度为50℃时其比活力最大。推测GSTd1参与了柑橘大实蝇嗅觉系统中气味信号物质的降解过程。

中文关键词:柑橘大实蝇谷胱甘肽S-转移酶原核表达蛋白纯化酶活力

Cloning, prokaryotic expression of GSTd1 gene from Chinese citrus fruit fly Bactrocera minax and the enzymatic characteristics analysis of the recombinant GSTd1 protein

Author Name	Affiliation	E-mail
Liu Yi	Institute of Entomology, College of Agriculture, Yangtze University, Jingzhou 434025, Hubei Province, China
Tian Xiaoli	College of Life Sciences, Yangtze University, Jingzhou 434025, Hubei Province, China
Mao Yongna	Jingzhou Branch of Hubei Academy of Forestry, Jingzhou 434020, Hubei Province, China
Gui Lianyou	Institute of Entomology, College of Agriculture, Yangtze University, Jingzhou 434025, Hubei Province, China
Wang Fulian	Institute of Entomology, College of Agriculture, Yangtze University, Jingzhou 434025, Hubei Province, China
Zhang Guohui	Institute of Entomology, College of Agriculture, Yangtze University, Jingzhou 434025, Hubei Province, China	zhangguohuiji@163.com

Abstract:To study the odor degradation mechanism in the olfactory system of Chinese citrus fruit fly Bactrocera minax, a glutathione S-transferase (GST) gene from the head transcriptome data was identified. Bioinformatic analysis of this gene was also performed. Real-time fluorescent quantitative PCR was used to investigate the tissue expression pattern of the gene in adult B. minax. Subsequently, this gene was cloned and expressed in Escherichia coli, and Western blot was used to detect the recombinant protein and the enzymatic characteristics of the protein were determined. The results indicated that a GST gene with a complete open reading frame (ORF), named GSTd1, was successfully cloned. The ORF of GSTd1 was 624 bp in length, encoding 207 amino acid residues. The predicted protein had a molecular weight of 23.72 kD and an isoelectric point of 6.16. GSTd1 from B. minax had no signal peptide and transmembrane domain, and thus it belonged to the cytoplasmic GSTs. Sequence alignment and phylogenetic analysis indicated that it belonged to the insect-specific GST Delta subfamily. It had the highest expression level in the antennae of B. minax adults. It exhibited a good catalytic activity for 1-chloro-2,4-dinitrobenzene (CDNB), and its maximum velocity was 11.76 μmol·mg^-1·min^-1 and and Michaelis constant was 0.224 mmol/L. The highest activity of GSTd1 from B. minax was detected under the conditions of pH 7.5 and 50 ℃. These results suggest that GSTd1 might participate in the degradation of odorant signal substances in the olfactory system of B. minax.

keywords:Bactrocera minax glutathione S-transferase prokaryotic expression protein purification enzyme activity

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