DNA条形码揭示日本纺织娘(Mecopoda niponensis)个体内和个体间的序列变异  

DNA Barcodes Reveal Intra-and Inter-Individual Sequence Variation in the Bush Cricket Mecopoda niponensis(Orthoptera :Tettigoniidae)

作　　者：周志军[1] 尚娜[2] 常岩林[1] 石福明[1] 

机构地区：[1]河北大学生命科学学院,保定071002 [2]河北大学图书馆,保定071002

出　　处：《生物技术通报》2014年第5期129-136,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31101632);教育部高等学校博士学科点专项科研基金项目(20101301120006)

摘　　要：采用DNA条形码通用引物对分布于我国的两种纺织娘14个个体进行了扩增与测定。结果显示,6个纺织娘Mecopoda elongata个体均可由PCR产物直接测序法获得良好测序结果,序列长度均为658 bp,且不存在碱基插入/缺失。而8个日本纺织娘M.niponensis个体PCR法直接测序结果峰图杂乱,软件无法识别,后经克隆法测定的85条克隆序列长度为580-662bp,其中,20条为明显的线粒体假基因(Nuclear sequences of mitochondrial origin,numts),包括4条存在终止密码子,15条存在碱基插入/缺失,1条存在读码框摆动;38条为疑似numts(含≥2个非同义替换位点)。利用Kimura 2-parameter(K2P)模型计算遗传距离发现,两种纺织娘之间的遗传距离为0.077,纺织娘及日本纺织娘个体间的遗传距离分别为0-0.008及0.030-0.051,而日本纺织娘个体内克隆间的遗传距离则分别为0-0.143(含numts)及0-0.083(不含numts)。邻接法系统发育树聚类结果显示,纺织娘形成一个单系分支,自举值为99,而日本纺织娘的COI和numts序列则聚类形成多个分支,提示这些numts序列可能是线粒体COI多次向核内转移的结果。The mitochondrial CO1 barcoding region of 14 individuals from two Mecopoda species with Folmer primers were amplified. All M. elongata COl fragments were 658 bp in length, without stop codons or indels. Due to the serious sequence ambiguities caused by direct sequencing, 85 clones were sequenced from eight M. niponertsis individuals, with sequence lengths ranging from 580 to 662 bp. Among these, 20 of the clones were distinct nuclear sequences of mitoehondrial origin, numts （ four clones with stop codons, 15 clones with indels, one clone was frameshift ） , and 38 clones contained point mutations that caused two or more amino acid sequence differences as compared with the orthologs. The Kimura 2-parameter （ K2P ） model was adopted to calculate genetic distance, and the results indicated that the genetic distances among M. elongata individuals varied from 0 to 0.008. Among M. niponensis, intra-individual clones ranged from 0 to 0.143 （ inclusive of numts ） or 0 to 0.083 （ exclusive of numts ） , and the inter-individual distance ranged from 0.030 to 0.051 （ exclusive of numts ） . The genetic distance between the two Mecopoda species was 0.077. Moreover, the neighbor-joining （ NJ ） phylogenetic tree based on the Kimura 2-parameter distances indicated that all the M. elongata individuals grouped together with high confidence （ 99% ） , and the real CO1 sequences and numts of M. niponensis clustered into several groups. These results indicate that numerous independent nuclear integration events in M. niponensis were scattered at various time points, which suggests a continuous process of numt generation.

关 键 词：纺织娘 线粒体DNA DNA条形码 线粒体假基因 异质性 

分 类 号：Q78[生物学—分子生物学]