马亿,张文兵,张宏玲,马红悦,韩海斌,庞保平,谭瑶,2022,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[J].环境昆虫学报,(5):1252-1263
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)
  
DOI:
中文关键词:  牧草盲蝽  细胞色素P450  钠离子通道  表达谱  序列分析
英文关键词:Lygus pratensis  cytochrome P450  sodium channel  expression profiling  sequence analysis
基金项目: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)
作者单位
马亿,张文兵,张宏玲,马红悦,韩海斌,庞保平,谭瑶 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 
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中文摘要:
      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2 003 bp,开放阅读框1 503 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6 072 bp,编码2 024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R14、R6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。
英文摘要:
      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 2 003 bp, with an open reading frame of 1 503 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 6 072 bp, encoding 2 024 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>5th instar nymph>4th instar nymph>3rd instar nymph>2nd instar nymph>1st 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 resistant and susceptible strains. In conclusion, over-expression of the P450 CYP6A13 gene might confer lambda-cyhalothrin resistance in L. pratensis. Our study provided a necessary basis for further studying the function of CYP6A13 involving in pyrethroid resistance mechanism of L. pratensis.
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