基于COⅠ基因的储粮象甲害虫的分子检测技术研究

Molecular detection of sitophilus oryzae,sitophilus zeamais and sitophilus granarius(Coleoptera:Curculionidae)based on mitochondrial COⅠgene

米象、玉米象和谷象是粮食与饲料储藏期间容易发生的3种常见象甲类害虫,仅从形态上进行鉴别存在一定的缺陷,建立快速准确鉴定3种常见储粮象甲类害虫的分子方法,可以弥补形态鉴别存在的不足。以3种常见储粮象甲害虫共38个地理来源样本为研究对象,采用DNA条形码技术,通过对线粒体细胞色素C氧化酶亚基(COⅠ)基因的特定区段(约426 bp)的测序和比对,以MEGA软件的K2-P双参数模型计算种内及种间遗传距离,以邻接法(NJ)构建系统发育树。碱基含量组成符合昆虫线粒体基因碱基组成的基本特征;种内遗传距离为0.000~0.007,平均为0.0034;种间遗传距离为0.163~0.207,平均为0.1932。邻接法系统发育树显示,聚类分析与形态学鉴定结果一致,3种储粮象甲各自聚为同一小支,且分支自展值均为100%,置信度高(98%),该段序列能有效区分3种储粮象甲。说明DNA条形码可以作为3种储粮象甲种间鉴别的分子方法。

Three major pest species of the genus Sitophilus(Coleoptera:Curculionidae),namely Sitophilus oryzae,S.zeamais and S.granarius,are of economic importance as they can cause significant damage to stored grains and food products.The use of morphological characteristics in weevil identification often displays shortcomings.This study aimed to explore molecular methods for identifying and distinguishing the three species of weevils.Thirty-seven Sitophilus pest samples were collected from 5 different countries,and were identified using DNA barcoding technique based on mtDNA COⅠgene.The partial fragments(426 bp)of COⅠgene were amplified using DNA barcoding universal primers.The obtained COⅠgene fragments were sequenced and aligned.The intra-and inter-species genetic distances were calculated using MEGA software according to the Kimura 2-parameter model.The phylogenetic tree was constructed using neighbor-joining method.Base composition and variation analyses showed the GC content was similar to the GC content observed in other insect COⅠgenes.Genetic distance analyzed by Kimura 2-parameter model showed that the interspecies genetic distance varied from 0.000 to 0.0034 while intraspecies genetic distance varied from 0.163 to 0.207.Phylogenetic tree was built with 98%confidence levels based on COⅠgene sequence.Same species of weevils were grouped within the same clade with bootstrapping value of 100%.Hence DNA barcoding can be used to distinguish between different Sitophilus species.