库蠓核糖体DNA内转录间隔区1序列的测定与分析

Sequence and analysis of rDNA-ITS1 of the genus Culicoides(diptera:ceratopogonidae)

目的测定核糖体DNA内转录间隔区1(nuclear ribosomal DNA internal transcribed spacer 1,rDNA-ITS1)序列进行库蠓种类鉴别与系统发育分析,以探究rDNA-ITS1序列在库蠓分子鉴定和系统发育研究方面的适用性。方法采用DNA扩增、纯化、克隆及测序的方法获得荒川库蠓Culicoides arakawai、凹缘库蠓C.holcus、连斑库蠓C.jacobsoni、印度库蠓C.indianus、霍飞库蠓C.huffi和肩宏库蠓C.humeralis等6种库蠓的rDNA-ITS1序列,基于Kimura 2-parameter公式计算种内和种间遗传距离,应用MEGA 6.06软件分析DNA序列碱基组成并以环纹埃蠓Allohelea annulata为外群构建系统发育树(邻接法和最大似然法)。结果上述6种库蠓的rDNA-ITS1序列经过Clustal W比对及人工校对和编辑后的长度为352bp,其中T、C、A、G 4种碱基的含量分别为36.8%、13.0%、30.0%和20.1%,A+T的含量(66.8%)高于C+G的含量(33.1%);K-2p遗传距离在种内和种间具显著差异(t=32.430,P<0.05);系统发育树中不同种类库蠓各自构成单系(群),同种类不同地理种群聚为一支,其与形态学鉴定结果一致。结论本研究证实了rDNA-ITS1序列可用于进行库蠓及其近似种的分子鉴定和系统发育分析。

We analysed the phylogenetic relationship and establish molecular identification of Culicoides species by sequencing the nuclear ribosomal DNA internal transcribed spacer 1(ITS1)sequences.The ITS1 sequences of six closely related species of the genus Culicoides,C.arakawai,C.holcus,C.jacobsoni,C.indianus,C.huffi and C.humeralis,were obtained by DNA sequencing.The calculation of genetic distance was based on Kimura 2-parameter formula.The base composition of ITS1 sequences and construction of phylogenetic trees which took the Allohelea annulate as outgroup were analyzed by MEGA 6.06v.Approximately 352 bp of ITS1 sequences were sequenced for those 6 species and the content of T,C,A,G of the sequences were 36.8%,13.0%,30.0%,20.1%,respectively.Statistical analysis of genetic distances showed that intraspecific genetic distances was different from that of interspecies significantly(t=32.430,P<0.05).The results of phylogenetic relationship were consistent with the morphological identification which indicated that the topologies of Maximum Likelihood(ML)tree and Neighbor-Joining(NJ)tree were quite similar;distinguished between different species of Culicoides clearly and no cross between different species;the same species gathered closely with each other.This study confirmed that rDNA-ITS1 sequences can be used for molecular identification and phylogenetic analysis in the genus Culicoides.