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橡实象虫(Curculio arakawai)是象甲科中危害柞树种子和嫩芽的重要害虫。为探讨利用线粒体细胞色素C氧化酶亚基Ⅰ(COⅠ)基因的特定区段作为DNA条形码快速准确识别橡实象虫的可行性,克隆了3个橡实象虫线粒体COⅠ基因5’端长度约556 bp的片段(GenBank登录号:JN258957,JN258958,JN258959),并与鞘翅目、鳞翅目、双翅目共24种昆虫的同源片段进行碱基组成多样性和系统进化分析。序列分析结果显示:橡实象虫与其它24种昆虫的COⅠ基因序列呈现丰富的遗传多样性,碱基位点的变异率为48.2%,序列平均差异为19.7%;COⅠ基因序列的AT含量(69.9%)明显高于GC含量(30.1%),存在显著的AT使用倾向性;碱基替换模式颠换大于转换,碱基替换数与物种两两遗传距离呈明显的线性关系;COⅠ基因序列的碱基变异速率较快的位点主要集中在序列中间区域的210～390 bp。基于COⅠ基因序列构建的NJ、ME和MP系统进化树均显示橡实象虫与鞘翅目象甲科(Curculionidae)昆虫的进化关系最近,聚在一起形成一个分支,鳞翅目等其它昆虫聚在一起形成另外一个分支;橡实象虫与鞘翅目象甲科Kyklioacalles属昆虫间遗传距离最小,与鳞翅目丝角蝶科Macrosoma sp.属昆虫间的遗传距离最大。根据COⅠ基因序列的多样性,能将橡实象虫明显地与其它昆虫区分开。
Curculio arakawai is an important pest that damages the seeds and shoots of oak trees in the family Coleoptera. In order to explore the feasibility of using the specific segment of the mitochondrial cytochrome C oxidase subunit Ⅰ (COⅠ) gene as a DNA barcode for rapid and accurate identification of Acorus elenophase, the length of the 5 ’end of COⅠ gene of three Acacia elegans was cloned 556 bp fragments (GenBank accession numbers: JN258957, JN258958, JN258959) were amplified by polymerase chain reaction (PCR), and their nucleotide diversity and phylogenetic analysis were compared with that of 24 coleopterans from Coleoptera, Lepidoptera and Diptera. The results of sequence analysis showed that the COⅠ gene sequences of Eucryptoporora and other 24 kinds of insects showed rich genetic diversity with a mutation rate of 48.2% at base loci and an average sequence difference of 19.7%. AT content of COⅠ gene sequence ( 69.9%) was significantly higher than that of GC (30.1%), and there was a significant tendency of AT to be used. The transversion of base substitution was greater than that of transversion, and the number of base substitutions was significantly linear with the pairwise genetic distance of species. The sites with faster mutation rate were mainly located in the region of 210-390 bp in the middle region of the sequence. The phylogenetic trees of NJ, ME and MP based on the COⅠ gene sequence showed that the evolutionary relationships between Acorus elemene and Coleoptera, such as Curculionidae, were the closest, clustered together to form a branch and other insects such as Lepidoptera clustered Together with the other branch. The genetic distance between the genus Kyklioacalles and the genus Kyklioacalles is the smallest, which is the largest among the insects of Macrosoma sp. According to the diversity of COⅠ gene sequences, the elephant worm could be clearly distinguished from other insects.