Zhang Yujie, Chen Wenlong, Li Ming, Yang Lin, Chen Xiangsheng
Institute of Entomology, Guizhou University, Guiyang, China.
Guizhou Key Laboratory for Plant Pest Management of Mountainous Region, Guizhou University, Guiyang, China.
PeerJ. 2019 Sep 6;7:e7641. doi: 10.7717/peerj.7641. eCollection 2019.
Due to the misuse of chemical pesticides, small green leafhoppers ( Matsuda) have developed resistance to pesticides, thereby posing a serious problem to the tea industry. Glutathione S-transferases (GSTs) are an important family of enzymes that are involved in pesticide resistance in Matsuda. GST sigma 1 (, GenBank: MK443501) is a member of the GST family. In this study, the full-length cDNA of was cloned by reverse transcription polymerase chain reaction (qPCR), and its taxonomic identity was examined. Furthermore, we performed bioinformatics and phylogenetic analyses of the gene and structural and functional domain prediction of the protein. The results demonstrate that EoGSTS1 belongs to the Sigma family of GSTs; the full-length cDNA is 841 bp with a 624-bp coding region that encodes a 23.68932-kDa protein containing 207 amino acids. The theoretical isoelectric point (IEP) was calculated to be 6.00. Phylogenetic analysis indicates that EoGSTS1 is closely related to the subfamily of the superfamily in order , whereas it is distantly related to of order . Amino acid sequence alignment of EoGSTS1 and GSTs from four other insects of order revealed protein sequence conservation. Tertiary structure analysis and structural domain functional predictions of the protein revealed that EoGSTS1 contains nine helices and two sheets with one conserved GST domain. The results of enzyme activity assay showed that recombinant (rEoGSTs1) protein had catalytic activity for substrate 1-chloro-2,4-dinitrobenzene (CDNB) and exhibited the highest activity at pH 7 and 25 °C. The Michaelis constant Km of rEoGSTs1 protein was 0.07782 ± 0.01990 mmol/L, and the maximum reaction rate Vmax was 12.15 ± 1.673 µmol/min⋅mg. Our study clarified the taxonomic identity of small green leafhopper and revealed some properties of the gene and its encoded protein sequence. According to the catalytic activity of the rEoGSTs1 enzyme on the model substrate CDNB, we infer that it functions in the degradation of exogenous substances.
由于化学农药的滥用，小绿叶蝉（松田氏）已对农药产生抗性，从而给茶叶产业带来严重问题。谷胱甘肽S-转移酶（GSTs）是一类重要的酶家族，参与小绿叶蝉的抗药性。GST sigma 1（，GenBank：MK443501）是GST家族的成员。在本研究中，通过逆转录聚合酶链反应（qPCR）克隆了其全长cDNA，并对其分类身份进行了检测。此外，我们对该基因进行了生物信息学和系统发育分析，并对该蛋白质进行了结构和功能域预测。结果表明，EoGSTS1属于GSTs的Sigma家族；全长cDNA为841 bp，编码区为624 bp，编码一个含有207个氨基酸、分子量为23.68932 kDa的蛋白质。理论等电点（IEP）计算为6.00。系统发育分析表明，EoGSTS1与目下的超家族亚家族密切相关，而与目的远缘相关。EoGSTS1与其他四种目昆虫的GSTs的氨基酸序列比对显示出蛋白质序列保守性。对该蛋白质的三级结构分析和结构域功能预测表明，EoGSTS1包含9个α螺旋和2个β折叠，具有一个保守的GST结构域。酶活性测定结果表明，重组（rEoGSTs1）蛋白对底物1-氯-2,4-二硝基苯（CDNB）具有催化活性，在pH 7和25℃时活性最高。rEoGSTs1蛋白的米氏常数Km为0.07782±0.01990 mmol/L，最大反应速率Vmax为12.15±1.673 μmol/min·mg。我们的研究阐明了小绿叶蝉的分类身份，揭示了该基因及其编码蛋白质序列的一些特性。根据rEoGSTs1酶对模型底物CDNB的催化活性，我们推断它在外源物质降解中起作用。
