单 位：
贵阳学院生物与环境工程学院贵州省山地珍稀动物与经济昆虫重点实验室;贵州大学烟草学院;贵州大学昆虫研究所贵州山地农业病虫害重点实验室;贵州中烟工业有限责任公司
关键词：
烟草甲;谷胱甘肽S-转移酶;基因表达;甲酸乙酯;RNA干扰
摘 要：
【目的】探究烟草甲Lasioderma serricorne谷胱甘肽S-转移酶(glutathione S-transferase, GST)基因LsGSTe1的分子特性和生物学功能。【方法】在烟草甲转录组数据的基础上,利用RT-PCR技术扩增LsGSTe1基因,并进行生物信息学分析;采用qPCR技术检测LsGSTe1在烟草甲不同发育阶段(低龄幼虫、高龄幼虫、蛹、低龄成虫、高龄成虫)及高龄幼虫不同组织(表皮、中肠、脂肪体、马氏管)中的表达水平,以及在甲酸乙酯熏蒸胁迫后的5龄幼虫中的表达变化。进一步采用RNAi技术沉默烟草甲5龄幼虫LsGSTe1基因,通过生物测定分析烟草甲对熏蒸剂甲酸乙酯的敏感性变化。【结果】获得LsGSTe1基因的全长cDNA序列(GenBank登录号:MN480468),开放阅读框长684 bp,编码227个氨基酸,N端和C端均存在催化保守位点。系统发育分析表明该基因属于GSTs的Epsilon家族。qPCR结果表明,LsGSTe1在烟草甲不同发育阶段均有表达,且在高龄幼虫期的表达量较高;表达部位主要在幼虫脂肪体,其次为中肠和表皮,而在马氏管中的表达量最低。LC_(30)(10μL/L)和LC_(50)(20μL/L)浓度的甲酸乙酯对烟草甲5龄幼虫熏蒸胁迫后,LsGSTe1的表达水平显著升高,分别是对照组的2.96和5.80倍。RNAi结果发现,RNAi 48 h和72 h后,LsGSTe1的表达量分别下降了79.9%和83.0%,沉默效率显著;RNAi 72 h后,dsLsGSTe1注射组与对照(dsGFP组)相比,LC_(50)浓度的甲酸乙酯处理5龄幼虫的死亡率明显提高了32.4%。【结论】推测LsGSTe1基因可能参与了烟草甲对甲酸乙酯的代谢与解毒过程。
译 名：
Expression of glutathione S-transferase gene LsGSTe1 and its relationship with ethyl formate tolerance in the cigarette beetle, Lasioderma serricorne (Coleoptera: Anobiidae)
作 者：
YAN Yi;XU Kang-Kang;YANG Hong;HU Da-Ming;YANG Wen-Jia;Provincial Key Laboratory for Agricultural Pest Management of Mountainous Regions, Institute of Entomology, Guizhou University;Guizhou Provincial Key Laboratory for Rare Animal and Economic Insect of the Mountainous Region, College of Biology and Environmental Engineering, Guiyang University;College of Tobacco Science, Guizhou University;China Tobacco Guizhou Industrial Co., Ltd.;
关键词：
Lasioderma serricorne;;glutathione S-transferase;;gene expression;;ethyl formate;;RNA interference
摘 要：
【Aim】 To explore the molecular characteristics and biological function of the glutathione S-transferase(GST) gene LsGSTe1 from the cigarette beetle, Lasioderma serricorne. 【Methods】 Based on the transcriptome database of L. serricorne, the full-length cDNA of LsGSTe1 was cloned using RT-PCR and then subjected to bioinformatics analysis. The expression levels of LsGSTe1 in different developmental stages(early instar larva, late instar larva, pupa, callow adult, and mature adult) and different tissues(integument, midgut, fat body, and Malpighian tubules) of the late instar larvae of L. serricorne, and the changes in the expression level of LsGSTe1 in the 5 th instar larvae after exposure to ethyl formate fumigation were detected via qPCR. The target gene LsGSTe1 in the 5 th instar larvae of L. serricorne was further knocked down by RNAi, and the changes in the susceptibility of the larvae to ethyl formate fumigation were determined by insecticide bioassay. 【Results】 The full-length cDNA sequence of LsGSTe1 in L. serricorne was cloned and deposited at GenBank under the accession number MN480468. The open reading frame of LsGSTe1 is 684 bp in length encoding a 227-amino-acid protein. LsGSTe1 has the conserved catalytic sites at the N-terminal domain and C-terminal domain. Phylogenetic analysis revealed that LsGSTe1 belongs to the Epsilon family of GSTs. The qPCR results showed that LsGSTe1 was constitutively expressed in the tested developmental stages and had a relatively higher expression level in the late instar larva. Tissue-specific expression profiles showed that the expression level of LsGSTe1 was the highest in the fat body, followed by in the midgut and integument, and the lowest in Malpighian tubules. The expression level of LsGSTe1 in the 5 th instar larvae subjected to fumigation treatment with LC_(30)(10 μL/L) and LC_(50)(20 μL/L) of ethyl formate was significantly increased by 2.96 and 5.80 times as high as that of the control, respectively. RNAi results showed that the expression levels of LsGSTe1 were significantly reduced by 79.9% and 83.0% at 48 and 72 h after RNAi, respectively. The mortality of the 5 th instar larvae treated with the LC_(50) of ethyl formate in the dsLsGSTe1 injection group at 72 h after RNAi increased by 32.4% as compared with that of the control group(dsGFP injection group). 【Conclusion】 It is inferred that LsGSTe1 may be involved in the detoxification of ethyl formate in L. serricorne.
