粘虫CYP6AE88和CYP332A1基因的鉴定和生物信息学分析  

Identification and bioinformatics analyses of genes of CYP6AE88 and CYP332A1 in Mythimna separata

作　　者：李敏[1,2] 李生才[2] 王青[1] 张虎芳[2] 

机构地区：[1]太原师范学院生物系,山西太原030031 [2]山西农业大学农学院,山西太谷030801

出　　处：《山西农业大学学报（自然科学版）》2016年第3期191-200,218,共11页Journal of Shanxi Agricultural University(Natural Science Edition)

基　　金：中国博士后研究经费(134845);国家自然科学基金项目(31501840);山西省科技攻关项目(NO.20150311010-7);太原师范学院大学生创新创业项目(CXCY1610)

摘　　要：鉴定粘虫Mythimna separata CYP450家族基因,分析其序列特征并推测可能的功能,为研究粘虫杀虫剂抗性分子机制提供理论依据。以棉铃虫Helicoverpa armigera CYP6AE20v2和CYP332A1作为询问序列,通过双向Blast方法分别检索粘虫转录组中CYP6AE和CYP332AcDNA序列,通过生物信息学的方法预测基因所编码的蛋白质序列的理化性质、疏水性、跨膜区、结构域、3D结构等以及可能的功能,并采用最大似然法（Maximum Likelihood,ML）分析夜蛾科代表物种的CYP6AE和CYP332A亚家族序列的系统发育关系。从粘虫转录组测序数据中鉴定出CYP6AE和CYP332A两个亚家族的基因,分别命名为CYP6AE88（GenBank登录号：KU145393）和CYP332A1（GenBank登录号：KU145394）。cDNA序列分析显示,CYP6AE88和CYP332A1的全长分别为1 713bp和1 815bp,分别编码509和503个氨基酸。蛋白质序列基本性质分析显示：CYP6AE88蛋白理论相对分子质量为58.9kD,等电点是7.95;CYP332A1蛋白理论相对分子质量为58.4kD,等电点是8.03。CYP6AE88和CYP332A1分别在5-22位和5-23位氨基酸之间有一个典型的疏水性区域;分别在2~21位和2~24位氨基酸之间形成一个典型的跨膜区。两个蛋白均不存在信号肽,亚细胞定位均为细胞质。蛋白质结构域分析分析显示：两个蛋白均含P450特有的5个特征序列,并且含有多个酶结合位点。3D结构分析显示,CYP6AE88含有α螺旋的数目是18条和β折叠数目是9股,CYP332A1含有α螺旋的数目是19条和β折叠10股。CYP6AE的系统发育分析显示：粘虫CYP6AE88与（草地贪夜蛾CYP6AE43＋海灰翅夜蛾CYP6AE49）聚为一支（BP=72%）,然后再与棉铃虫CYP6AE20v2/50/19形成单系（BP=100%）;CYP332A的聚类分析显示：粘虫CYP332A1与棉铃虫CYP332A1聚为一支,bootstrap值为100%。研究结果为进一步研究粘虫CYP6AE88和CYP332A1基因杀虫剂抗性分子机制奠定了基础。To identify CYP450 genes of Mythimna separate, analyze their bioinformatic characteristics and infer the possible functions for further research on their roles in insecticide resistance. CYP6AE88 and CYP332A1 of M. separata were retrieved from its transcriptome annotation data and identified with bidirectional Blast using Helicoverpa armigera CYP6AE2Ov2 and CYP332A1 as query, respectively. The structure, characteristics and possible functions of CYP6AE88 and CYPgg2A1 genes were analyzed using bioinformation methods. The phylogenetic relationships of CYP6AE88 and CYP332A1 sequences of M. separate and representative noctuid species was constructed using maximum likelihood methods, respectively. Two CYP450 genes were identified from M. separata transcriptome sequenced data and named as CYP6AE88 （GenBank accession number： KU145393） and CYP332A1 （GenBank accession number： KU145394）, respectively. They were 1590 bp and 1542 bp in length,encoding 509 and 503 amino acids, respectively.Basic properties of the protein CYP6AE88 and CYP332A1 showed that the molecular weight was 58.9kD and 58.4 kD, the isoelectric point was 7.95 and 8.03, the predicted hydrophobic region was 5-22 and 5-23, the transmembrane region was 2-21 and 2-24, respectively. Both of the two deduced amino acid sequences had no signal peptide sequence and were localized in cytoplasm. Protein domain prediction showed that the encoded proteins of these two genes contained five P450 characteristic sequences and had multiple enzyme active sites. The 3D structural prediction showed that CYP6AE88 had 18 a-helix and 9 13-strands, while CYP332A1 had 19 a-helix and 10 l]-strands. Phylogenetic analysis of CYP6AE showed that M. separate CYP6AE88 were grouped with （Spodoptera frugiperda CYP6AE43 -- Spodopt era littoralis CYP6AE49） （BP = 72%）, and then formed monophyletie relationship with Helicoverpa armigera CYP6AE20v2/50/19 （BP= 100%）. Clustering analysis of CYP332A showed that M. separate CYP332A1 and H. armigera CYP332A1 were the first clus

关 键 词：粘虫 CYP450 CYP6AE88 CYP332A1 生物信息学分析 

分 类 号：S433.4[农业科学—农业昆虫与害虫防治]