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.
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
沈中元
唐顺明
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)
出处
《蚕业科学》
CAS
CSCD
北大核心
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)。
关键词
家蚕追寄蝇
GST1-1
基因克隆
酶学特性
活性分析
Exorista sorbillans Wiedemann
GST1-1
Gene cloning
Enzymatic property
Activity analysis