6种鱼mtDNA D-loop及其邻近区段的序列比较分析

The sequence comparison of mtDNA D-loop and adjacent regions in six fish species

采用PCR技术对乌克兰鳞鲤Cyprinus carpio、鲫Carassis auratus、鲢Hypophthalmichthys molitrix、鳙Aristichthys nobilis、草鱼Ctenopharyngodon idellus和乌苏里拟鲿Pseudobagrus ussuriensis共28个个体(分属于天津市动植物抗性重点实验室应用PCR-RFLP方法已检测出的不同单倍型)的mtDNA D-loop及其邻近区段进行扩增并测序,获得了大小为1 500～1 800 bp的扩增产物。用Clustal 1.83软件与GenBank中的鲤、金鱼、鲢、草鱼、长吻鮠5种鱼的mtDNA全序列进行排序比较,确定扩增产物包含完整的tRNAPro、D-loop、tRNAPhe序列以及12S rRNA长度约为400 bp的部分序列。将6种鱼共28个样本的序列提交GenBank后获得序列号为KC292921～KC292948。运用Mega 4.0软件计算出6种鱼的碱基组成和碱基差异,基于Kimura双参数模型计算种间的遗传距离,并与GenBank中5种鱼的mtDNA序列一起构建NJ系统树。序列结构分析表明,在6种鱼序列的4个区段中,D-loop区段在种内、种间的差异性均高于另外3个区段。6种鱼NJ系统树的结果与传统分类方法一致,可为鱼类分类以及种类鉴定提供科学依据。另外,6种鱼各种单倍型均在所测得序列中找到相应的酶切位点,印证了RFLP实验结果,在酶切位点以外也发现了同种鱼的不同单倍型间存在碱基差异。

The mtDNA D-loop and its adjacent regions were amplified and sequenced in 28 individuals from different haplotypes detected using PCR-RFLP in Ukraine scale carp（ Cyprinus carpio）, crucian carp（ Carassius auratus）, silver carp （Hypophthalmichthys molitrix）, big head earp（Aristichthys nobilis）, grass carp （Ctenopharyngodon idellus） and Ussuri bullhead（Pseudobgrus ussuriensis） by PCR. The amplified fragment length of mtDNA D- loop and its adjacent regions in the fish were varied from 1 500 bp to 1 800 bp, and are compared with the mtDNA complete sequences of five fish species from GenBank by Clustal 1.83 software. It was found that the amplified fragment was comprised of complete tRNA^Pro, D-loop, tRNA^Pro sequences and about 400 bp part of the 12S rRNA sequence. Sequences of 28 individuals were submitted to GenBank for serial numbers （KC292921- KC292948 ）, and the base composition and difference of the six species, and the genetic distance were calculated based on Kimura＇s 2-Parameter model and NJ phylogenetic tree with five sequences from GenBank was constructed by Mega 4.0 software. The sequence structure analysis showed that the intraspecific and interspecific differences in D-loop region were higher in the four region sequences of six species than those in other species. NJ phylogenetic trees of six species were consistent with the traditional classification methods, showing useful for fish species classification and identification. In addition, restriction sites of all kinds of haplotypes were found in sequences, which is consistent with PCR-RFLP experimental results. More variation out of the restriction sites were observed compared with RFLP analysis.