锈色粒肩天牛幼虫的COⅠ、COⅡ、Cytb和28S基因片段序列分析

Sequence analysis of COⅠ, COⅡ, Cytb and 28S gene fragments from Apriona swainsoni larvae

【目的】基于分子生物学手段,探讨可快速准确鉴别锈色粒肩天牛Apriona swainsoni幼虫与其他天牛幼虫的目标基因,为该保健昆虫的食药用安全提供保障。【方法】利用线粒体COⅠ、COⅡ、Cytb基因和28S细胞核基因的通用引物分别克隆获得4种基因片段,并进行同源序列检索、多重比对、序列信息分析及进化树构建。【结果】基因克隆最终获得锈色粒肩天牛4种基因序列的片段大小分别为817、545、434和1088 bp。特殊位点分布结果显示:28S的保守位点占比最高,其后依次为Cytb、COⅠ和COⅡ,变异率则正好相反。COⅠ、COⅡ和Cytb基因片段的碱基组成和替换信息特点均表现为A+T含量>G+C含量,颠换高于转换,28S基因片段则表现为A+T含量<G+C含量,颠换低于转换。序列信息分析结果表明:线粒体基因和核基因的序列结构存在差异,核基因较线粒体基因更为保守。4种基因序列的分子进化树均能将锈色粒肩天牛幼虫与桑天牛A.germari等其他天牛幼虫区分开。【结论】COⅠ、COⅡ、Cytb和28S基因均可作为鉴定该虫的分子生物学参考依据。

[Objective]Based on molecular biological methods,this research was intended to explore the the target genes that could identify Apriona swainsoni larvae from other Cerambycidae larvae,so as to provide a guarantee for the food and drug use of A.swainsonias larvae.[Method]Four different gene fragments were cloned using the general primers of COⅠ,COⅡ,Cytb and 28S genes before the homologous sequence retrieval,multiple comparison,sequence analysis and molecular phylogenetic tree construction were carried out.[Result](1)The fragments of the four gene sequences obtained by gene cloning were 817,545,434 and 1088 bp,respectively.(2)In light of the distribution of special loci,28S had the highest proportion of conserved sites,followed by Cytb,COⅠand COⅡ,while the variability rate was just the opposite.The base composition and replacement of the COⅠ,COⅡand Cytb gene fragments were all shown as A+T contents>G+C contents,with the rate of translocation higher than that of the conversion.However,when it came to the 28S gene fragment,it was A+T cotents<G+C cotents,with the rate of translocation lower than that of the conversion.(3)There existed differences in the sequence structure between mitochondrial genes and nuclear genes with the latter more conservative than the former.(4)The molecular phylogenetic relationship based on those four gene sequences could distinguish A.swainsoni larvae from other Cerambyeidae larvae,such as A.germari.[Conclusion]COⅠ,COⅡ,Cytb and 28S genes could be used as the molecular reference for the identification of A.swainsoni larvae.