深圳市白纹伊蚊rDNA ITS2区克隆及SNP分析

Cloning and SNP analysis of rDNA ITS2 of Aedes albopictus in Shenzhen

目的克隆和序列分析深圳市白纹伊蚊核糖体DNA(rDNA)第2内转录间隔区(ITS2),并探明其rDNA ITS2核酸的特异性及不同个体在rDNA ITS2区的单核甘酸的多态性(Single Nuclear Polymorphism,SNP),以便利用ITS2基因的高度保守性及其蚊媒不种群在ITS2片段的多态性来分子鉴别白纹伊蚊。方法PCR特异扩增白纹伊蚊rDNA ITS2,利用QIAquick技术从琼脂糖胶上回收纯化扩增的目的ITS2 DNA片段,纯化后的目的ITS2 DNA片段被连接到TA载体上,在含有青霉素的琼脂糖胶平面上筛选含目的ITS2 DNA片段的阳性克隆,阳性克隆经含有青霉素的LB液体培养基培养,用Ultrapure^(TM)质粒提取试剂盒提取克隆质粒,用双酶切鉴定插入的片段大小,DNA序列分析仪分析每个蚊rDNA ITS2的序列,用生物信息系统进行白纹伊蚊rDNA ITS2 SNP的差异分析。结果518bp全长的深圳市白纹伊蚊rDNA ITS2被克隆和序列分析,深圳市四个不同地理的白纹伊蚊rDNA ITS2的同一性为97％,而两个地方之间的比较有99％的同一性,其rDNA ITS2基因单核苷酸存在转换、颠换和缺失,在518bp的范围内有6个C→T,2个A→G的转换,3A→T,一个A→C的颠换,3个CG和一个AC缺失。结论白纹伊蚊rDNA ITS2有高度的保守性,不同个体也表现了单核苷酸的多态性。

Objective To clone and sequence analyse rDNA ITS2 ofAedes albopictus and investigate speciality and Single Nuclear Polymorphism of rDNA ITS2 of Aedes albopictus in Sbenzhen so as to identify different species of mosquito using rDNA ITS2 gene. Methods rDNA ITS2 of Aedes albopictus was amplified using PCR. PCR products were electrophoresed in 1.5 % agarose gel, and extracted with a gel extraction kit. ITS2 fragment purified was linked to TA- vector Among the obtained transformants, six clones beating the correct target were selected by the precise size of the inserts. Sequence analysis were performed for those clones using DNA Sequencer. Sequence homology search was done with BLAST program to the Genbank database. Results 518bp full fragment of rDNA ITS2 of Aedes albopictu was cloned and sequenced. ITS2 of Aedes albopictu of four different isolates from Shenzhen showed 97% homogeny, and 99% homogeny was indicated between two different Aedes albopictu.s from Shenzhen. Single Nuclear Polymorphism of rDNA ITS2 of Aedes albopictus showed transition, transversion and deletion. There were six C→T, two A→G transitions, threeA→T, one A→C transversions and three CG, one AC deletions. Conclusion rDNA ITS2 gene of Aedes albopictu.s have high homogeny, but SNP was also observed among deffrent Aedes albopictu.s.