牧草盲蝽CYP6A13和钠离子通道LPVSSC基因的克隆及参与高效氯氟氰菊酯的抗性分析  

Cloning of CYP6A13 and sodium channel gene LPVSSC and their involvement in analysis on the resistance of lambda-cyhalothrin in Lygus pratensis(Hemiptera∶Miridae)

作　　者：马亿 张文兵 张宏玲 马红悦 韩海斌[2] 庞保平 谭瑶 MA Yi;ZHANG Wen-Bing;ZHANG Hong-Ling;MA Hong-Yue;HAN Hai-Bin;PANG Bao-Ping;TAN Yao(Research Center for Grassland Entomology,Inner Mongolia Agricultural University,Hohhot 010020,China;Institute of Grassland Research of Chinese Academy of Agricultural Science,Hohhot 010010,China)

机构地区：[1]内蒙古农业大学草原昆虫研究中心,呼和浩特010020 [2]中国农业科学院草原研究所,呼和浩特010010

出　　处：《环境昆虫学报》2022年第5期1252-1263,共12页Journal of Environmental Entomology

基　　金：国家自然科学基金(32160651,31560519);内蒙古农业大学高层次人才科研启动基金(NDYB2019-02)。

摘　　要：昆虫对拟除虫菊酯类杀虫剂产生抗性主要是解毒酶活性的增强和钠离子通道的敏感度降低所致。本研究采用RT-PCR和RACE技术克隆获得了牧草盲蝽Lygus pratensis P450 CYP6A13基因(GenBank登录号:MN782520)及钠离子通道LPVSSC基因cDNA全长序列(GenBank登录号:MW821485)。其中CYP6A13基因全长2003 bp,开放阅读框1503 bp,编码500个氨基酸,预测蛋白分子质量为57.2 kDa,主要位于细胞内质网,等电点为5.77,无跨膜区和信号肽,存在血红素结合保守功能区。同源比对与系统进化分析表明,牧草盲蝽的CYP6A13与分类学关系上较为接近的昆虫同源蛋白间高度相似。钠离子通道序列LPVSSC开放阅读框6072 bp,编码2024个氨基酸,预测蛋白分子质量为228.94 kDa,等电点为4.99,无信号肽,有多个跨膜区域,4个同源结构域(I~IV),每个结构域有6个跨膜片段(S1~S6),存在MFM基序。基因表达谱结果表明:CYP6A13在牧草盲蝽成虫头部的表达量高于胸、腹部;室内筛选的高效氯氟氰菊酯抗性R 14、R 6品系的CYP6A13表达量是敏感品系的18.74和5.45倍;CYP6A13表达量在雌、雄成虫之间无显著差异;不同发育期基因表达量为:初羽化成虫>5龄若虫>4龄若虫>3龄若虫>2龄若虫>1龄若虫。测序比对高效氯氟氰菊酯抗性与敏感品系的钠离子通道序列IIS4~IIS6区域,未发现存在可能导致靶标抗性的氨基酸突变。结果表明CYP6A13基因过表达可能导致了牧草盲蝽对高效氯氟氰菊酯的抗药性,为深入解析CYP6A13基因介导抗药性的功能研究奠定基础。The pyrethroid resistance of insects is mainly caused by the increased activity of detoxification enzymes and the decreased sensitivity of sodium ion channels.The full-length sequences of P450 CYP6A13 of Lygus pratensis(GenBank accession number:MN782520)and the sodium channel LPVSSC(GenBank accession number:MW821485)cDNAs were cloned by using RT-PCR and RACE technology.The full length of CYP6A13 gene was 2003 bp,with an open reading frame of 1503 bp,encoding 500 amino acids.The predicted molecular weight of the protein was 57.2 kDa,the isoelectric point(pI)was 5.77,and there was no transmembrane region and signal peptide.The protein was predicted to be located in the endoplasmic reticulum with heme binding conserved functional regions.Homologous comparison and phylogenetic analysis showed that CYP6A13 was highly similar to the homologous proteins of insects,which were relatively similar in taxonomy.The open reading frame of sodium ion channel sequence LPVSSC was 6072 bp,encoding 2024 amino acids,and the predicted molecular weight of the protein was 228.94 kDa with the isoelectric point(pI)of 4.99.There were multiple transmembrane regions without signal peptide,including four homologous domains(I~IV),each having six transmembrane segments(S1~S6),and the MFM motif exists.The gene expression results showed that the expression level of CYP6A13 in the head was higher than that in the thorax and abdomen;the expression of CYP6A13 in the lambda-cyhalothrin resistant strain R14、R6 were 18.74 and 5.45 fold higher than that in the susceptible strain;there was no significant difference between male and female adults on the expression level of CYP6A13;the gene expression of CYP6A13 in different development stages ranked as:newly emerged adult>5 th instar nymph>4 th instar nymph>3 rd instar nymph>2 nd instar nymph>1 st instar nymph.Sequencing analysis results showed that no amino acid mutations involved in the lambda-cyhalothrin resistance were found in the IIS4~IIS6 domain of the sodium channel sequence between the resistan

关 键 词：牧草盲蝽 细胞色素P450 钠离子通道 表达谱 序列分析 

分 类 号：Q963[生物学—昆虫学] Q965.9[农业科学—农业昆虫与害虫防治] S433[农业科学—植物保护]