小地老虎谷胱甘肽-S-转移酶AiGSTs1的分子特性及其对杀虫剂胁迫的响应
Molecular characterization of glutathione-S-transferase (AiGSTs1) in Agrotis ipsilon subject to insecticide stress
李茂业 黄 岩 蒋秀云 何佶弦 刘 苏
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DOI：10.7679/j.issn.2095-1353.2021.092
作者单位：作物有害生物综合治理安徽省重点实验室，植物病虫害生物学与绿色防控安徽普通高校重点实验室， 安徽农业大学植物保护学院，合肥 230036；四川省烟草公司广元市公司，广元 628017
中文关键词：小地老虎；谷胱甘肽-S-转移酶；原核表达；毒死蜱；高效氯氟氰菊酯；解毒代谢
英文关键词：Agrotis ipsilon; GST; expression; chlorpyrifos; lambda-cyhalothrin; metabolic detoxification
中文摘要：
【目的】 鉴定小地老虎Agrotis ipsilon sigma家族谷胱甘肽-S-转移酶（GST）基因，明确其编码蛋白质的催化活性，阐明该基因在小地老虎不同发育期、不同组织及在毒死蜱和高效氯氟氰菊酯胁迫下的表达模式，为深入探究小地老虎GST在杀虫剂解毒代谢中的功能提供理论基础。【方法】 利用同源检索方法从小地老虎转录组中鉴定GST基因，利用原核表达系统表达重组蛋白并使用试剂盒检测其活性，使用实时荧光定量PCR技术分析基因的表达模式。【结果】 从小地老虎转录组中鉴定了一个编码sigma家族GST的cDNA序列，命名为AiGSTs1。其编码的AiGSTs1蛋白含有谷胱甘肽结合位点和底物结合位点，具有GSTs的典型特征。在大肠杆菌中成功表达了重组AiGSTs1蛋白，测得此蛋白不仅具有谷胱甘肽转移酶活性，还具有过氧化物酶活性。毒死蜱和高效氯氟氰菊酯均可抑制重组AiGSTs1蛋白的活性。AiGSTs1在供试的小地老虎不同发育期和组织中均有表达，但在蛹期和幼虫脂肪体中表达量最高。使用LD50剂量毒死蜱和高效氯氟氰菊酯处理小地老虎幼虫后，AiGSTs1的表达水平显著上升。【结论】 本研究明确了小地老虎AiGSTs1的序列、所编码蛋白的活性和表达模式。毒死蜱和高效氯氟氰菊酯能够诱导AiGSTs1表达水平上调，表明该基因可能在这2种杀虫剂的解毒代谢中起重要作用。
英文摘要：
[Objectives] To provide a theoretical basis for exploring
the role of GSTs in insecticide detoxification in Agrotis ipsilon by identifying the sigma class glutathione-S-transferase (GST) gene in A. ipsilon,
determining the catalytic activity of the protein encoded by this gene, and
elucidating its expression patterns in different developmental stages, tissues,
and in response to the stress induced by chlorpyrifos and lambda-cyhalothrin. [Methods] The A.
ipsilon GST gene was identified
from transcriptome data using a homological search method. Recombinant protein
was expressed in a prokaryotic expression system and its activity detected with
commercial kits. The expression pattern of the gene was analyzed using reverse
transcription quantitative PCR. [Results] A cDNA (designated AiGSTs1) sequence encoding a putative
sigma class GST was identified in the A.
ipsilon transcriptome. Protein encoded by AiGSTs1 contain glutathione-binding and substrate-binding sites,
which are typical characteristics of GSTs. Recombinant AiGSTs1 protein was
expressed in Escherichia coli, and
this protein displayed not only GST activity but also peroxidase activity. In
addition, the activity of AiGSTs1 was significantly inhibited by chlorpyrifos
and lambda-cyhalothrin. AiGSTs1 mRNA
was detectable in different developmental stages and tissues. Highest
expression was in the pupal stage and the larval fat body. Furthermore, AiGSTs1 transcription levels were
significantly upregulated in larvae exposed to LD50 doses of
chlorpyrifos and lambda-cyhalothrin. [Conclusion]
Transcription of AiGSTs1 can be induced by chlorpyrifos
and lambda-cyhalothrin, which suggests that the AiGSTs1 gene may play essential roles in the metabolic detoxification
of these insecticides.