核桃举肢蛾线粒体CO I和Cytb基因片段序列分析

核桃举肢蛾（Atrijuglans hetaohei）是危害核桃果实的主要害虫，严重影响核桃的产量和商品价值。在最新的形态学分类上，核桃举肢蛾属于鳞翅目、麦蛾总科、黑展足蛾属。通过同源序列比对探讨利用DNA条形码技术（DNA barcoding）对核桃举肢蛾进行分类鉴定的准确性。运用2对通用引物分别扩增核桃举肢蛾线粒体细胞色素C氧化酶亚基I（CO I）基因和细胞色素b（Cytb）基因片段，并与鳞翅目麦蛾总科、巢蛾总科、凤蝶总科、螟蛾总科等昆虫的同源片段进行碱基组成多样性和系统进化分析，扩增得到核桃举肢蛾CO I基因664 bp片段和Cytb基因479 bp片段。结果表明:核桃举肢蛾线粒体CO I基因片段中，A、T、G和C 4种核苷酸的含量分别为29.5%、39.4%、15.1%和16.1%，A+T的含量(68.9%)明显高于G+C含量（31.1%），存在显著的AT使用倾向性。在Cytb基因片段中， A、T、G和C 4种核苷酸的含量分别为33.4%、41.5%、10.2%和14.8%，A+T的含量为74.9%，也明显高于G+C含量（26%）。两段序列都以T-A间颠换和T-C间转换为主要替换方式，且密码子第2位点保守性最强，第3位点变异率最高。基于CO I和Cytb基因片段构建的NJ系统进化树均显示核桃举肢蛾与麦蛾总科昆虫处于同一个分支之下。利用DNA条形码技术得到的核桃举肢蛾分子生物学分类结果与传统的形态学分类结果一致。

Sequence Analysis of the Fragments of CO I and Cytb Genes from Atrijuglans hetaohei

Atrijuglans hetaohei Yang is the main pest of walnut fruits,affecting the yield and the commercial value of walnut seriously.In the latest morphological classification,A.hetaohei belongs to Lepidoptera,Gelechioidea,Atrijuglans.In order to investigate the accuracy of A.hetaohei identification by the method of DNA barcoding technology,we used homologous sequence alignment and analysis.Mitochondrial cytochrome C oxidase subunit I (CO I) gene and cytochrome b (Cytb) gene fragments were amplified with two pairs of universal primers,and subsequently were analyzed on their base composition diversity and pholygenesis with homologous fragments of Gelechioidea,Yponomeutoidea,Papilionoidea,Pyraloidea,etc.Nucleotide sequences of 644 bp for CO I gene and 479 bp for Cytb gene were obtained,respectively.The result of sequence analysis showed that in CO I gene sequence of A.hetaohei,the contents of A,T,G and C nucleotides were 29.5%,39.4%,15.1% and 16.1%,respectively,and the content of A+T (68.9%) was obviously higher than that of G+C (31.1%),indicating a strong AT bias.The result of sequence analysis in Cytb gene sequence of A.hetaohei showed that the contents of A,T,G and C nucleotides were 33.4%,41.5%,10.2% and 14.8%,respectively,and the content of A+T was 74.9%,which was also obviously higher than that of G+C (26%).In these two sequences,the main base substitution mode was T-A transversion and T-C transition,and the second codon was the most conservative site,while the third codon had the highest mutation rate.The constructed NJ phylogenetic trees based on CO I and Cytb gene sequences showed that A.hetaohei and Gelechioidea insects formed a monophyletic clade.The classification result of A.hetaohei by means of DNA barcoding technology is in accordance with the traditional morphological classification.