二种土甲族幼虫的分子鉴定  

Studies on molecular identification of two larvae of Opatrini

作　　者：张建英[1] 李彪 贾龙[2] 丁荣荣 Zhang Jianying;Li Biao;Jia Long;Ding Rongrong(School of Life Sciences,Ningxia University,Yinchuan 750021,China;School of Agriculture,Ningxia University,Yinchuan 750021,China)

机构地区：[1]宁夏大学生命科学学院,宁夏银川750021 [2]宁夏大学农学院,宁夏银川750021

出　　处：《农业科学研究》2021年第3期10-16,71,共8页Journal of Agricultural Sciences

基　　金：宁夏自然科学基金资助项目“宁夏土甲族昆虫优势种类幼期分子鉴定研究——基于DNA条形码技术”(2019AAC03013)。

摘　　要：土甲族(Opatrini)是拟步甲科(Tenebrionidae)中较大的族之一,其昆虫是主要的农、林、牧业及仓储害虫。土甲族幼虫取食作物根部,对作物为害严重,然而由于其幼虫属间种间差异小,难以从形态上进行准确鉴定。以土甲族粗背伪坚土甲(Scleropatrum horridum horridum)和阿笨土甲(Penthicus alashanica)为研究对象,制成粗背伪坚土甲雌性成虫、雄性成虫、低龄幼虫、高龄幼虫和阿笨土甲雌性成虫、雄性成虫、低龄幼虫、高龄幼虫8个样品,分别提取8个样品的基因组DNA,再通过PCR技术扩增每个样品的COⅠ基因和16S基因序列,并分别进行同种及不同种COⅠ基因和16S基因序列比对分析,结果显示:粗背伪坚土甲和阿笨土甲16S基因序列相似性分别为100%、98.97%~99.56%,COⅠ基因序列相似性分别为98.49%~99.50%、98.63%~99.24%。种内最大差异均小于限值(2%),种间最小差异也明显高于限值(2%)。由结果可知:同种土甲成、幼虫相同基因序列的相似性均大于98%,分子鉴定结果与形态学鉴定结果一致,说明采用线粒体16S基因和COⅠ基因同时作为DNA条形编码的标记基因进行幼虫鉴定是可行的。本研究结果不但提供了一种鉴定幼虫的新方法,且获得的序列可用于粗背伪坚土甲和阿笨土甲各虫态的分子鉴定。DNA条形码技术可以弥补传统分类学在幼期标本鉴定上的短板,且快速、准确,可为虫害防治提供基础数据。Opatrini is one of the largest families of Tenebrionidae,which is a major agricultural,forestry,animal husbandry and storage pest.The larvae feed on the roots of crops and do serious harm to the crops,it is difficult to identify it accurately in morphology,and a new molecular technique is urgently needed to solve the problem.In this study,Scleropatrum horridum horridum and Penthicus alashanica were used as experimental subjects,eight samples including the two Opatrini insects’female adult,male adult,young larvae,old larvae were prepared and the genomic DNA of 8 samples were extracted respectively,then the COⅠgene and 16S gene sequences of each sample were amplified by PCR,and the COⅠgene and 16S gene sequences of the same species and different species were compared and analyzed,the results showed that the similarity of 16S gene sequence of the two Opatrini insects were 100%and 98.97%~99.56%,the similarity of COⅠgene sequence of the two Opatrini insects were 98.49%~99.50%and 98.63%~99.24%.The maximum intra-species difference was less than the limit(2%),and the minimum inter-species difference was significantly higher than the limit(2%),The results showed that the similarity of the two gene sequences of the adults and larvae of the two species was more than 98%.The results of molecular identification were consistent with those of morphological identification.It is feasible to use mitochondrial 16S gene and COⅠgene as marker genes of DNA strip coding for larval identification.The results of this study not only provide a new method for larval identification,but also can be used for the molecular identification of the different states of the two beetles.DNA bar code technology can make up for the shortcomings of traditional taxonomy in identification of larvae stage specimens,and provide basic data for pest control in time.

关 键 词：土甲族 COⅠ基因 DNA条形码 快速鉴定 

分 类 号：S433[农业科学—农业昆虫与害虫防治]