Mitochondrial ATP synthase F0 subunit 8(ATP8) and subunit 6(ATP6) gene in muscular tissue of Asian black bear Sichuan subspecies(Ursus thibetanus mupinensis) are amplified for the first time through PCR technology with primers designed according to ATP8 genes and ATP6 genes of some other mammals reported ,which are cloned,sequenced and analyzed preliminarily.The result shows that the full length of the PCR product is 942bp which contains ORF 842 encoding the ATP8 and ATP6 genes with a 43bp overlap between them.The ATP8 sequence is 204bp encoding a protein with 67 amino acid residues whose molecular weight is 7.9KD and isoelectric point is 10.35.The ATP6 gene is 682bp encoding a protein with 226 amino acid residues whose molecular weight is 24.8KD and isoelectric point is10.63.Both of them are highly homologous to those of other mammals reported.Construction of phylogenetic tree based on nucleotide sequence manifests that the Asian black bears with Sichuan subspecies have closer relationship to the American black bears.

By means of the primer designed according to the conservative sequence of mammalian mitochondrial gene,mitochondrial 12S rRNA and 16S rRNA gene were amplified from the Asian black bear Sichuan subspecies' muscle tissue total DNA by PCR method.Their sequences were determined and analyzed.The result indicated that 12S rRNA gene length and 16S rRNA gene length of Sichuan black bear were 965 bp and 1 580 bp respectively.The further sequence analysis showed that 12S rRNA and 16S rRNA gene of Sichuan black bear had a higher evolution rate,which had more differences than that of American black bear,brown bear,polar bear,spectacled bear and panda,but Sichuan black bear had more homology with American black bear.

Mitochondrial Cytochrome C Oxidase subunit Ⅲ gene(mtCOXⅢ) in muscular tissue of Asiatic black bear Sichuan subspecies(Ursus thibetanus mupinensis) was amplified for the first time using PCR technology with primers designed according to mtCOXⅢ genes of some mammals reported,which was cloned,sequenced and analyzed preliminarily.The mtCOXⅢ sequence of Asiatic black bear Sichuan subspecies is 784 bp containing ORF 783 bp and codes 261 amino acid residues whose isoelectric point is 6.47 and molecular weight is 29840.6 Da.The mtCOXⅢ gene is highly homologous to some mammals reported.Further analysis indicates that the position,the type and the quantity of the function sites of cloned mitochondrial Cytochrome C Oxidase subunit Ⅲ are almost same to some mammals reported.

Primers were designed based on the NADH-ubiquinone oxidoreductase chain 1 gene DNA sequences (ND1) of several species. The total DNA extracted from the hair follicle tissue of the Asian black bear Sichuan subspecies (Ursus thibetanus ssp.mupinensis) was amplified by PCR to obtain the ND1 gene sequence. The softwares GenScan, Blast 2 1, ORF finder and Clustal W were utilized in DNA sequence prediction, similarity comparison, ORF search and multi-sequence alignment, respectively, and the software PredictProtein was used in protein prediction and structure analysis. The result showed that the ND1 gene sequence is 957bp long and contains a 957 nucleotide ORFs (open reading frames) encoding 317 amino acid residues. The amino acid sequence had a pI of 7.83 and a molecular weight of 35.6×103 Da.The DNA sequence of ND1 gene of Asian black bear of Sichuan and the amino acid sequence encoded by it shared relatively high similarity with the DNA sequence and amino acid sequence of other bear species, raccoon (Procyon) and Ailurus fulgens.

Mitochondrial ATP synthase (F1Fo-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In the present study, using RT-PCR combined with in silico cloning, we isolated and sequenced the cDNA encoding the inhibitor protein of the giant panda (Ailuropoda melanoleuca). The deduced protein sequence showed that the protein is composed of 106 amino acids and the estimated molecular weight of the ATPIF(1) protein is 12.32 kDa with an isoelectric point (pI) of 10.17. Alignment analysis revealed that the deduced protein sequence shares 66%, 78.3%, 66%, 72.6%, 77.4%, and 78.3% homology with that of Mus musculus, Pan troglodytes, Rattus norvegicus, Bos taurus, Macaca mulatta, and Homo sapiens, respectively. Topology prediction showed that there are three protein kinase C phosphorylation sites, one amidation site, three N-myristoylation sites, one casein kinase II phosphorylation site, and one tyrosine kinase phosphorylation site in the ATPase inhibitor. In particular, amino acids in the region between 39 and 72, which is the minimum sequence showing ATPase inhibitory activity, were highly conserved in the protein.

In order to provide scientific material for promulgating the giant panda(Ailuropoda melanoleuca)nucleus gene inheritance characteristic,for the formulation gene protection countermeasure and to put resources to rational use,the expression sequence of ubiquinol-cytochrome c reductase complex ubiquinone-binding protein(QP-C)gene from the giant panda was analyzed.The result indicates that the cDNA sequence length is 335 bp and contains a 246-nucleotide open reading frame encoding 81 amino acid residues.The pI of the protein is 10.50 and its mole cular weight is 9.60×103 Da.Topology prediction shows these QP-C proteins contain a N-glycosylation site,a potential protein kinase C phosphorylation site and a Casein kinase II phosphorylation site.Comparing the cDNA sequence with amino acid sequence of giant panda's QP-C gene and cDNA sequence with the amino acid sequence of other species' QP-C gene shows high similarity.The similarity is 82.3% to 95.2% and 81.5% to 95.1%.

The fragment of mitochondrial genome 764 bp of Sichuan black bear has been cloned and determined.According to homologous comparision,the DNA fragment contains two protein-coding genes(ND3 and ND4L) and one tRNA-Arg gene.The DNA sequence with amino acid sequence of black bear's ND3 and ND4L gene and the DNA sequence with amino acid sequence of Malay bear',brown bear',American black bear' and polar bear' ND3 and ND4L gene have the high homology,the homology is 90% and 95%,93% and 97%,91% and 96%,93% and 97%,respeclively;89% and 98.98%,88% and 98.98%,89% and 100%,89% and 98.98%,respeclively.The homology of tRNA-Arg gene with brown bear、American black bear and polar bear' is 95.65%、94.20% and 94.20%,respeclively.The analysis of topology indicates that ND3 gene of Asian black bear has a less Protein kinase C phosphorylation site than American black bear's.

The cDNA fragment of ribosomal protein L26 (RPL26) was cloned from Ailuropoda melanoleuca using RT-PCR method. The cDNA fragment is composed of 475bp, containing an open reading frame of 145 amino acids. Alignment analyses indicated that the nucleotide sequence and the deduced amino acid sequence showed high identity to other known RPL26 sequences from vertebrates and invertebrates. The cDNA sequence was used to construct phylogenetic trees with other known vertebrate and invertebrate RPL26 sequences, and the obtained trees demonstrated similar topology with the classical systematics, indicating the potential value of RPL26 gene in phylogenetic analysis.

Objective To explore the features of the cytochrome c oxidase subunit Ⅰ (COXⅠ) of Asian black bear Sichuan subspecies. Methods Primers were designed according published gene sequences of other bear species. A DNA sequence was amplified by PCR from the bear Sichuan subspecies’s hair-follicle tissue total DNA, then the PCR product was sequenced. The results were analyzed and a forecast analysis was carried on. Results The amplified COXⅠ gene of Sichuan black bear was 1 605 bp and contained a 1545-nucleotide open reading frame encoding 514 amino acid residues. The overall base composition of COXⅠ gene is 26.9 %, 23.1%, 18.3 %, and 31.8 % respectively to A, C, G, and T. The start codon of the COXⅠ is ATG, while the termination codon TAA. The protein’s pI is 6.29 and weight is 57.2×10 3 . The similarity analysis and phylogenetic tree analysis indicated that Sichuan black bear has closer relationship to American black bear, but not to brown bear and polar bear. Among dogs, caws and rats, it is closer to the former one. Conclusion There are high similarities in the DNA sequence, protein amino acid sequence of Asian black bear Sichuan subspecies with other bear species, indicating a high conservativity of COXⅠ in these involved species.

Objective To investigate the sequence characteristic of black bear's mitochondrial NADH-ubiquinone oxidoreductase chain 4 gene(ND4).Methods Primers were designed according to certain species' NADH-ubiquinone oxidoreductase ND4 in which the DNA sequence had been reported.The DNA sequence was amplified by PCR from the Asian black bear Sichuan subspecies's(Ursus thibetanus mupinensis)hair-follicle tissue total DNA and were analyzed with its amino acid sequence.Results The ND4 gene sequence length was 1 402 bp and contained a 1 377-nucleotide open reading frame encoding 459 amino acid residues.The amino acid sequence's pI was 9.37 and weight was 51.5×10 3.The DNA sequence and corresponding amino acid sequence of black bear's ND4 gene had the high similarity with those of other mammalian animals' ND4 gene,and the similarity was 89% to 91% and 94% to 95%.The result of phylogenetic tree was basically consistent with the traditional species system phylogenetic relations.Topology prediction showed the Asian black bear Sichuan subspecies's ND4 proteins contained N-glycosylation site,kinase C phosphorylation sites,casein kinase Ⅱ phosphorylation site,N-myristoylation site and leucine-rich region.In addition,black bear's ND4 protein contained leucine zipper pattern and Leucine-rich region.Conclusion The mammalia animal's ND4 gene has the high conservative nature.The pretein coded by ND4 gene has high uniformity in the function.

We obtained the complete mitochondrial genome of U.thibetanus mupinensis by DNA sequencing based on the PCR fragments of 18 primers we designed. The results indicate that the mtDNA is 16,868 bp in size, encodes 13 protein genes, 22 tRNA genes, and 2 rRNA genes, with an overall H-strand base composition of 31.2% A, 25.4% C, 15.5% G and 27.9% T. The sequence of the control region (CR) located between tRNA-Pro and tRNA-Phe is 1422 bp in size, consists of 8.43% of the whole genome, GC content is 51.9% and has a 6bp tandem repeat and two 10bp tandem repeats identified by using the Tandem Repeats Finder. U. thibetanus mupinensis mitochondrial genome shares high similarity with those of three other Ursidae: U. americanus (91.46%), U. arctos (89.25%) and U. maritimus (87.66%).