摘要
以白鲟科和鲟科的13种鲟鱼为研究对象,设计7对特异引物,利用PCR技术扩增鲟形目(Acipenseriformes)鱼类线粒体DNA(mtDNA),扩增片段大小在2～3kb,总长度约为整个mtDNA的97%。同时利用3个限制性内切酶,即MboⅠ、CfoⅠ和HaeⅢ对13种鲟形目鱼类进行限制性酶切片段长度多态性(RFLP)分析。结果总共检出233条电泳带,其中209条有多态现象,表明鲟形目鱼类mtDNA存在广泛变异。但是各个扩增片段的变异程度并不相同,其中NADH脱氢酶亚基基因的RFLP多态性较丰富,其次是细胞色素c氧化酶亚基基因,而两个rRNA基因最贫乏。说明在鲟形目鱼类mtDNA的进化中NADH脱氢酶亚基基因速率最快,细胞色素c氧化酶亚基基因次之,rRNA基因最慢。分子系统分析结果显示,鲟科的6种鱼与匙吻鲟和白鲟分成两大支;鲟科中两种鳇鱼没有聚到一起,支持了鳇鱼不能作独立分类单元的观点。本研究旨为鲟形目鱼类遗传和进化研究提供科学依据。
Acipenseriformes consists of 27 species, most of which are endangered. Although they have very high values in scientific research and economy, little is known about their phylogenetic relationship and evolution. In this research ,7 primer pairs were designed to amplify the sequences of mitochondrial DNA from the alcohol-preserved fin rays or muscles of eight species of Acipenseriformes by means of polymerase chain reaction technique .They are Chinese paddlefish (Psephurus gladius), paddlefish (Polyodon spathula), European huso sturgeon (Huso huso), Siberian huso sturgeon (H.dauricus), Chinese sturgeon (A.sinensis), Amur sturgeon (A.schrenckii), River sturgeon (A. dabryanus), sturgeon (A.baerii), Green Sturgeon(A.mediros), Shortnose sturgeon(A.breviro), Russian sturgeon(A.guelden), Adriatic sturgeon(A.naccarii)and Star sturgeon(A.stellatus).The fragments amplified by the 7 prime pairs were marked as seg1,seg2,seg3,seg4,seg5,seg6 and seg7,the lengths of which were 3?kb,2.6?kb,2?kb,2.4?kb,2.2?kb,2.5?kb and 2?kb, respectively. Their total length almost covered 97% of the complete sequences of the mitochondrial genome. The cleavage of seg4 by HaeⅢ produced 19 fragments,while CfoⅠ had no cleavage site on seg2.The numbers of DNA electrophoresis bands produced by each amplified fragment were as follows: 42 for seg1,24 for seg2,27 for seg3,47 for seg4,36 for seg5,33 for seg6 and 24 for seg7. All the segments had differences except seg2 and seg7,which were cut by CfoⅠ.From the data above,it can be seen that,seg4 obtained the most abundant DNA bands, and seg1 the second most, while the other segments obtained less without the inclusion of D-loop-containing seg6. This indicates that the genes encoding several subunits of NADH dehydrogenase (ND1, ND2, ND4, ND5 and ND6) have rather large variations in base number and higher rate of evolution, while those coding for the three subunits of cytochrome c oxidase (COⅠ, COⅡ, COⅢ) have rather small variations and slower rate. Of the 24 DNA electrophoresis bands obtained by the two rRNA genes in the eight species, only 14 showed differences, having the smallest variations. This indicates that rRNA genes are the most conservative in the evolution of mtDNA of Acipenseriformes.Of the eight species, the genetic distance between A.sinensis and A.dabryanus is 0.005?8,the smallest one, while that between P.gladius and A.naccari is 0.048?5, the largest one. The UPGMA molecular phylogenetic tree of the eight species constructed on the genetic distance was divided into two large branches: P.gladius and P.spathula formed a branch, whereas the other species another one. The latter was divided into two small ones: A.sinensis and A.dabryanus formed a small one, the other 4 sturgeon species another small one. However, H.huso and H.dauricus did not get together.This study supports the research results on sequence analysis of ND4L-ND4 genes, but does not conform with those of analysis of 12 S and 16 S rRNA gene sequences and part of cytochrome b gene sequence. Besides, our results support the conclusion from all the molecular data, i.e., the extant two Huso sturgeon species cannot be classified as a separate Huso. However, the genetic distance between P. gladius and P.spathula, which belong to two different genera, is smaller than that among the species of Acipenser. This may be relevant to the endonucleases used in this experiment. More endonucleases should have beed used so to provide more convenient conditions for researches on the genetic structures of mtDNA and sequencing the complete mt genome of Acipenseriformes.
出处
《中国水产科学》
CAS
CSCD
北大核心
2005年第4期383-389,共7页
Journal of Fishery Sciences of China
基金
国家基础工作重点专项(2002-2003)
中国水产科学研究院青年基金.
