重要蚊虫复合组DNA鉴定芯片靶基因的适用性分析

Applicability Analysis of DNA Identification Chip Target Genes for Important Mosquito Complex Groups

通过分析白纹伊蚊亚组、尖音库蚊复合组和杂鳞库蚊复合组蚊虫核糖体内转录间隔区Ⅱ(Internal transcribed spacer,ITS2)、细胞色素c氧化酶亚基Ⅰ(cytochrome c oxidase subunitⅠ,COⅠ)及Cytb细胞色素b氧化酶基因(cytochrome b,Cytb),为3组蚊虫DNA鉴定芯片研制提供理论依据。提取以上3组蚊虫基因组DNA,PCR扩增COⅠ、ITS2以及Cytb基因,分析3个目的基因在3组蚊虫DNA鉴定芯片研制中的适用性。结果显示共获得8种蚊虫COⅠ、Cytb及ITS2基因序列24条;碱基组成结果显示COⅠ、Cytb的A+T含量高达74%,均高于ITS2;遗传距离显示3组蚊虫复合组的组间进化分歧数均大于0.200;3个基因的系统进化树显示,复合组(亚组)的蚊虫序列聚成一大簇,且可信度均大于98,尖音库蚊复合组内的蚊虫序列无法与各对应的序列聚成一簇。得出结论 COⅠ、ITS2及Cytb基因可联合用于杂鳞库蚊复合组蚊虫、白纹伊蚊亚组及尖音库蚊复合组中部分蚊虫的基因芯片鉴定,为蚊虫复合组的鉴定提供新方向。

By analyzing the internal transcribed spacer region 2(ITS2), cytochrome oxidation subunit I(CO Ⅰ) and cytochrome b gene(Cytb) sequences of Aedes albopictus Subgroup, Culex vishnui Complex and Cx. pipiens Complex, the scientific foundation for the development of DNA identification chips for the three mosquitos complex(subgroup) was provided. PCR was used to amplify the sequences of CO Ⅰ, ITS2 and Cytb genes, the applicability of three target genes for DNA identification microarrays of three mosquito complex was analyzed. Results showed that 24 gene sequences of CO Ⅰ, Cytb and ITS2 were obtained from eight tested mosquito species. The A+T contents of CO Ⅰ and Cytb were 74%, which were greater than that of ITS2;the genetic distance revealed that the evolutionary divergence among the three mosquito complex was greater than 0.200;the phylogenetic tree of the 3genes showed that the mosquito sequences in the complex(subgroup) were clustered in a large branch and the reliability was greater than 98. The mosquito sequences in the Cx. pipiens Complex didn’t cluster with the corresponding sequence. This research showed that CO Ⅰ, ITS2 and Cytb genes could be used in the development of gene chip for the identification of mosquitoes in Cx. vishnui Complex, Ae. albopictus Subgroup and some mosquitoes of Cx. pipiens Complex, which provided a new direction for mosquito identification.