| In this research, 8 primer pairs were designed to amplify the sequences of mitochondrial DNA from the alcohol-preserved fin rays of Chinese paddlefish Psephurus gladius and Yangtze sturgeon Acipenser dabryanus by means of polymerase chain reaction technique. The PCR products were purified, and then used as template for direct sequencing using a primer walking strategy. The complete mtDNA sequences of the two fish were determined, and features of the sequences and molecular evolution were analyzed. The major research achievements and implications of this study were abstracted as following.1 The results of the PCRThe fragments amplified by the 8 prime pairs were marked as seg.1, seg.2, seg.3, seg.4, seg.5, seg.6, Seg.7, seg.8, the lengths of which are 3 kb, 2.6 kb, 2 kb, 2.4 kb, 2.2 kb, 2.5 kb, 2 kb, 0.3kb,respectively. Their total length covers the complete sequences of the mitochondrial genome.2 General features of Psephurus gladius and Acipenser dabryanus mtDNAThe entire nucleotide sequences of the L strand of Psephurus gladius and Adpenser dabryanus were identified 16483bp and 16437bp long respectively, both containing the same 2 rRNA genes 13 protein-coding genes, 22 tRNA genes and the putative control region (D-LOOP), All genes code with L strand except ND6 and 8 tRNA genes, which code with H strand, as found in other vertebrate. The base composition of the L strand of Psephurus gladius mtDNA is 30.5% A, 30.0% C, 15.7% G, 23.8% T. That of Adpenser dabryanus mtDNA is 30.2 % A, 29.7% C, 16.4% G, 23.7% T. Like other vertebrate, the mtDNAs composition is AT-rich. The nucleotide bias against G is particularly marked at third codon positions of protein-coding genes except ND6.while 12 protein-coding genes only below 10% of sites are G, 40.1% and 42.7% of G at third codon positions in ND6 genes of the two fish. tRNA genes arranged 67-75bp in sizes, and all can be folded into a cloverleafsecond structure with 5bp in the anticodon stems. In control regions, three conserved sequence blocks and tandem repeat sequences are found, like as other sturgeons. The two sequences are available in GenBank now, and the accession numbers are NC005834 and NC005451.3 Molecular evolution of mtDNA in Acipenseriformes12S rRNA, 16S rRNA ,tRNA ,13 protein-coding genes(except the start and stop codon) and amino acid sequences were used to reconstruct Maximum Parsimony and Neighbor Joining phylogenetic trees, respectively. Except the two 12S rRNA phylogenetic trees, 8 phylogenetic trees are supported the relationships in species of Acipenseriformes, but the protein-coding genes and amino acid sequence trees have the highest supporting degree. The ratios of transition-transversion are about 6.0,except the 16S rRNA gene, which is 2.52. It can be seen from the divergence and similarity of different genes that the protein-coding genes share the highest genetic divergence percent and have observable different values between family and genus. Genetic divergence percent is above 13.3 between different family, about 10 between different genus, and 4.5-8.2 between different species. Genetic divergence percent of other genes are one three that of protein-coding genes. The substitution rates of the mtDNA of Acipenseriformes were calculated, and the rates of tRNA,12S,16S,protein coding synonymous substitution (ks) and nonsynonymous substitution (ka) and amino acid are 0.0097-0.0129 X 10"9nuc7year.site,0.0086-0.0115 X 10"9nuc./year.site, 0.0112-0.0150X10"9 nuc/year.site, 1.420-1.893 X W9 nuc/year.site, 0.0072-0.0095X 109 nuc./ year, site and 0.0108-0.0144 X10'9 am./year.site. |
