中华按蚊代谢抗性相关基因多态性分析

Nucleotide polymorphism of genes related to metabolism and detoxification in Anopheles sinensis(Diptera:Culicidae)

目的测定中华按蚊现场样本的宏基因组,分析代谢解毒酶相关基因的多态性。方法在山东济宁曹县和北湖区用人帐诱捕采集按蚊,基于形态和分子特征鉴定确认蚊种,进行宏基因组Illumina测序。从NCBI数据库下载属于CYP450s、GSTs和Coe基因家族的中华按蚊8个代谢解毒酶基因序列。应用DNASTAR将宏基因组测序结果与代谢解毒酶相关基因序列进行对比,在SeqMan Pro软件上分别进行读取和SNP分析,根据开放阅读框确定SNP的位置,并判别其为同义突变SNP或非同义突变SNP。结果分子鉴定的山东济宁曹县和北湖区中华按蚊样本分别为31只和12只,分为2组进行宏基因组测序。Illumina测序每组样本产生的reads数近200 Mb,平均长度101 bp,GC含量平均为41%(曹县)和42%(北湖区),mapping到8个代谢解毒酶基因序列上的reads数为391~2810条,每个位点的平均覆盖度的范围为23.72~144.93。本研究中华按蚊8个代谢抗性基因共有135个SNP位点,其中109个导致同义突变,占80.74%,26个非同义突变SNP位点(19.26%);每Kb的SNPs位点数为8.73~29.94;非同义突变SNPs在密码子的位置主要是第1位,占50.00%(13/26),第2位和第3位分别占26.92%(7/26)和23.08%(6/26)。结论Illumina测序获得中华按蚊宏基因组序列中代谢抗性相关基因具有相当程度的多态性,可为进一步研究中华按蚊的抗性分子机制提供重要数据。

Objective To sequence the metagenome of field Anopheles sinensis samples,and analyze the polymorphism of genes related to metabolic detoxification enzyme.Methods Anophelesmosquitoes were captured with people as bait in Caoxian and Beihu,from Jining Shandong.An.sinensis species were identified by morphologic and molecular markers.The metagenome was carried out by Illumina platform.Eight genes of metabolic detoxification enzymes download from NCBI database were belong to CYP450 s,GSTs and Coe,respectively.Metagenomic reads was mapping to the gene sequences related to metabolic detoxification enzymes by DNASTAR software.The reads data collection and SNP analysis were performed by SeqMan Pro software,respectively.The position of SNPs in codons was fixed by open reading frame,and synonymous mutation SNPs or nonsynonymous mutation SNPs was determined.Results obtained from Caoxian and Beihu Jining Shandong.They were divided into 2 samples for metagenomic sequencing.The reads generated by each sample from Illumina sequencing was nearly 200 Mb.The mean length was 101 bp,and GC content were41%and 42%.The number of reads mapping to the 8 metabolic detoxification enzyme genes ranged from 391 to 2810 and mean coverage of each locus from 23.72 to 144.93.Total of 135 SNP positions in 8 metabolic detoxification enzyme genes was found in this An.sinensis population.Of which 109 lead to synonymous mutations,as 80.74%;and 26 non-synonymous mutation SNP positions,percentage for 19.26%.The range of SNP numbers per Kb was from 8.73 to 29.94.The position of nonsynonymous mutant SNPs in the codon was mainly the first,the percentage for 50.00%(13/26),the second and third position was for 26.92%(7/26)and 23.08%(6/26),respectively.Conclusion resistance genes with metagenome sequences of An.sinensis obtained by Illumina platform,which could provide important basis for further study of the molecular resistance mechanism.