家蚕追寄蝇谷胱甘肽硫转移酶GST1-1的特性分析及其在灭蚕蝇胁迫下的活性变化  

Characteristics of Glutathione S-Transferase1-1 from Exorista sorbillans and Analysis on Its Change in Activity Under Miecanying Stress

作　　者：王闪闪 王耒耒 陈诺婧依 Zufan Bekele Kassa 张彩红 王梅仙 朱娟 沈兴家[1,2] 沈中元 唐顺明[1,2] Wang Shanshan;Wang Leilei;Chen-Nuo Jingyi;Zufan Bekele Kassa;Zhang Caihong;Wang Meixian;Zhu Juan;Shen Xingjia;Shen Zhongyuan;Tang Shunming(Jiangsu Key Laboratory of Sericultural Biology and Biotechnology,School of Biotechnology,Jiangsu University of Science and Technology,Zhenjiang Jiangsu 212100,China;Key Laboratory of Silkworm and Mulberry Genetic Improvement,Ministry of Agriculture and Rural Affairs,Sericultural Research Institute,Chinese Academy of Agricultural Sciences,Zhen-jiang Jiangsu 212100,China)

机构地区：[1]江苏科技大学生物技术学院,江苏省蚕桑生物学与生物技术重点实验室,江苏镇江212100 [2]中国农业科学院蚕业研究所,农业农村部蚕桑与遗传改良重点实验室,江苏镇江212100

出　　处：《蚕业科学》2022年第3期198-208,共11页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金项目(32072791,31372376);国家蚕桑产业技术体系蚕用兽药研制团队项目(ARS-18-ZJ0303)。

摘　　要：家蚕追寄蝇(Exorista sorbillans Wiedemann)寄生家蚕幼虫导致蝇蛆病的发生。谷胱甘肽硫转移酶(glutathione S-transferase, GST)是一类多功能酶家族,对昆虫在解毒、杀虫剂抗性、氧化应激保护等方面起到重要的作用。本文以家蚕追寄蝇为实验材料,使用RACE技术,获得了家蚕追寄蝇谷胱甘肽硫转移酶基因的全长cDNA序列,将其命名为EsGST1-1(GenBank:OK104864)。EsGST1-1全长813 bp,包含一段627 bp完整开放阅读框(ORF),编码208个氨基酸残基,分子质量为23.58 kD,等电点为5.498,该蛋白属于GST的Delta类家族。序列比对结果显示,EsGST1-1与丝光绿蝇(Lucilia sericata)的GST基因亲缘关系最近。进一步检测了家蚕追寄蝇EsGST1-1基因在不同发育时期和不同组织的表达模式,结果显示在卵产后0 h、幼虫脂肪体和表皮、蛹期第6天以及雌雄成虫的腹部与足部中EsGST1-1高量表达。此外,利用原核体外诱导表达获得了EsGST1-1的重组蛋白。酶活试验结果显示EsGST1-1酶活性为0.820 4 U/mg。热稳定性实验结果显示EsGST1-1在30~40℃时活性最高,随温度的升高活性下降。对灭蚕蝇胁迫下的GST活性测定发现:杀虫活性呈剂量依赖效应;寄蝇谷胱甘肽硫转移酶活力与灭蚕蝇剂量呈负相关;EsGST1-1基因的表达量与灭蚕蝇剂量呈负相关。上述研究结果为进一步开发防治蝇蛆病新型药剂提供基础信息。Exorista sorbillans Wiedemann parasitizes silkworm larvae and causes myiasis. Glutathione S-transferases(GSTs) is a family of multifunctional enzymes, playing a vital role in detoxification, insecticide resistance and protection of oxidation stress for insects. In this study, E. sorbillans was used as the experimental material, and the full-length cDNA sequence of E. sorbillans glutathione S-transferases 1-1 gene named as EsGST1-1(GenBank: OK104864) was obtained by using RACE technology. With a total length of 813 bp, EsGST1-1 contains a 627 bp complete open reading frame(ORF) and encodes 208 amino acid residues. It has a molecular weight of 23.58 kD and an isoelectric point of 5.498, belonging to the Delta family of GSTs. The results of sequence alignment showed a closest relationship between EsGST1-1 gene and GST gene of Lucilia sericata. Subsequently, we further investigated the expression patterns of EsGST1-1 gene at different developmental stages and in different tissues of E. sorbillans. The results revealed that EsGST1-1 was predominantly expressed in eggs at 0 h after oviposition, in larval fat body and epidermis, on the sixth day of the pupal stage, and in the abdomen and legs of adults. In addition, the recombinant EsGST1-1 protein was obtained by prokaryotic induced expression in vitro. The enzyme activity of recombinant EsGST1-1 protein was 0.820 4 U/mg. The results of thermal stability assays showed that EsGST1-1 presented the highest activity at 30-40 ℃, which declined with the increase of temperature. The results of GST activity under Miecanying stress showed a dose-dependent manner. There was a negative correlation between the glutathione S-transferase activity and the dosage of Miecanying. The expression of the EsGST1-1 gene was negatively correlated with the dosage of Miecanying. The above results provide some information for developing new drugs on the silkworm myiasis in the future.

关 键 词：家蚕追寄蝇 GST1-1 基因克隆 酶学特性 活性分析 

分 类 号：S884.62[农业科学—特种经济动物饲养]