鲟形目鱼类rDNA扩增及限制性位点分析

Amplification and restriction sites analysis of rDNA in Acipenseriformes

使用长PCR技术扩增鲟形目(Acipenseriformes)15个种的核糖体DNA(rDNA)18S rRNA基因与28S rRNA基因之间约2.5 kb的片段,并用7个限制性内切酶对该片段进行限制性位点分析。结果显示,使用LATaqDNA聚合酶及GCBuffer结合长PCR技术能够很好地扩增鲟形目鱼类的rDNA。根据酶切图谱可确定93个限制性位点,其中21个为保守位点,72个多态信息位点。HhaⅠ酶切的带型在所有个体中完全一致,提示这些位点对保持rDNA的功能有重要作用。一些酶的酶切产物长度之和大于扩增产物的长度,暗示鲟形目鱼类个体内存在多个rDNA等位基因。对种间遗传距离进行计算,并构建UPGMA分子系统树,发现鲟科鱼类的系统关系与以往研究有较大差别,这可能是受个体内rDNA等位基因存在的影响,说明用酶切片段来重建鲟科鱼类的系统树不太可靠。文中还讨论了高GC含量模板的PCR扩增和rDNA在鲟形目分子系统关系中的使用。

The phylogenetic and evolutionary relationships within Acipenseriformes are of scientific significance, but views on it based on different data are also debated. In this paper, nuclear ribosomal DNA（rDNA） of two paddlefishes and thirteen sturgeons were amplified by polymerase chain reaction（PCR）, and nine restriction endonucleases（Afa Ⅰ , Ava Ⅱ , Dde Ⅰ , Hae Ⅲ, Hha Ⅰ , Mbo Ⅰ , MspⅠ , Nci Ⅰ, Xsp Ⅰ ） were used to digest the PCR products for testing the difference of restriction sites. The molecular phylogenetic tree of Acipenseriformes was also reconstructed based on genetic distance. To amplifiy rDNA effectively, LA Taq DNA polymerase, GC buffer（Takara, Dalian） and long PCR method were used,and the expected DNA segments of about 2.5 kb were obtained. After suitable enzyme reactions, two endonucleases（ Msp Ⅰ, Nci Ⅰ ） produced nondescript DNA electrophoresis bands which were excluded in analysis here. Six to nineteen restriction DNA bands were identified respectively for the other seven endonucleases. The total number of restriction DNA electrophoresis bands was ninety-three, twentyone of which were conservative and seventy-two were variable among the specimens of Acipenseriformes. I generated six bands from all the 15 species, indicating the restriction sites play important roles in active functions of rDNAs. Total sizes of electrophoresis bands produced by four endonucleases did not equal to the sizes of PCR products, implying multiple alleles of rDNA existed in an individual of Acipenseriformes. The presence of multiple alleles within individuals may be an evolutionary character,just like mtDNA het- eroplamy in sturgeons. The pair-wise genetic distances based on restriction sites ranged from 0.021 1（ Polyodon spathulaus Psephurus gladius ） to 0.145 3 （Polyodon spathulaus A. dabryanus ）. The UPGMA molecular phylogenetic tree of the fifteen species was divided into two large clusters--two paddlefishes forming a cluster, whereas the sturgeons forming the other one. It was the same results as the reported ones. The relationship among sturgeons may be obscured by the presence of multiple alleles, which had some difference from the other research results that A. baerii, and then A. axyrinchus was separated from the other species respectively. Future studies should focus on divergence investigation and allele types within and between individuals.