Cdna, Genomic Sequence, Overexpression And Related Research Of Crystallin Alpha-b Gene (cryab) Of The Giant Panda And Troponin C1 Gene (tnnc1) And The Giant Panda And The Black Bear

The Giant Panda (Ailuropoda melanoleuca) is a rare species currently found only in China. They are known as a "living fossil". The black bear (Ursus thibetanus mupinensis) is listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), the appendix I species; the National 2 levels of key protections wild animals; and China Red Data Book of Endangered Animals, V species.For many years, studies on the Giant Panda and Black Bear have been mainly concentrated on fields such as breeding and propagation, ecology, morphology, taxology, physiology and pathological biochemistry. Recently, researches on genetic diversity, parentage and phylogenesis et al. have been developed, while reports on functional gene are handful. This study was conducted at the molecular level.1,Primary content, aims and innovationThis study was conducted using RT-PCR technique and Touch-down technology to amplify the cDNA sequence and the genomic sequence of CRYAB gene and TNNC1 gene from the total RNAs and genomic DNA extracted from the skeleton muscle of the Giant Panda repectively, and then analyzed the sequence characteristics and compared it with those of human and other mammalian species reported. We also constructed the recombined expression plastid(pET28a) containing CRYAB and TNNC1 cDNA and transformed it into E. coli BL21 which was then induced to express the protein of interest. This study not only set forth characters of the cDNA and genomic sequence, but also the overexpression research lay a foundation for purifing and further studing the structure and the funtion of CRYAB and TNNC1 protein, which accumulate schemes and experiences for further developing the study at molecular levelAt present, studies on functional genes and their biological function are rare and there is not any report on the CRYAB gene and TNNC1 gene from the Giant Panda. We studied the Giant Panda and Black Bear, which are both the rarest wild animal, using the modern molecular technology and analyzed its sequence feature and overexpression. Therefore, this study is original paper. The achievement from this study can accumulate scheme and experiences for further developing studies on numbers of functional genes and their biological function. In addition, we can obtain significative information from the study to study genes encoding ribosomal protein subunits information from other mammals.2,Main results and conclusionsHere we have cloned the cDNA and the genomic sequence of CRYAB gene from the Giant Panda(Ailuropoda melanoleuca) and TNNC1 gene from the Giant Panda (Ailuropoda melanoleuca) and the Black Bear (Ursus thibetanus mupinensis) using RT-PCR technology and Touchdown-PCR, respectively.1. The length of cDNA fragment cloned contains an open reading frame of 528bp encoding 175 amino acids and the length of the genomic sequence is 3189bp, containing three exons and two introns. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other four species studied, including Homo sapiens, Mus musculus, Rattus norvegicus and Bos taurus. The homologies for nucleotide sequences of Giant Panda CRYAB to that of these species are 93.9%,91.5%,91.5% and 95.3%, respectively, and the homologies for amino acid sequences are 98.3%,97.1%,97.7% and 99.4%, respectively. Topology prediction shows that there are only four Casein kinaseâ…¡phosphorylation sites in the CRYAB protein of the Giant Panda. The Cdna of CRYAB was transfected into E. coli, and the CRYAB fused with the N-terminally His-tagged protein gave rise to the accumulation of an expected 24KDa polypeptide, which accorded with the predicted protein.2 The TNNC1 gene length of cDNA fragment of Giant Panda cloned contains an open reading frame of 486bp bp encoding 161 amino acids and the length of the genomic sequence is 2831bp, contains six exons and five introns. Cloned TNNC1 gene cDNA fragment of the Black Bear contains a 486bp encoding 161 amino acid open reading frame, the structure gene is 2758bp, also contains six exons and five introns. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence for TNNC1 gene of the two species are highly conserved to other 11 species have been reported. Topology prediction shows that there are five Casein kinaseâ…¡phosphorylation sites, one Protein kinase C phosphorylation site, one N-myristoylation site, three EF-hand calcium-binding domains and one N-glycosylation site in the TNNC1 protein of the Giant Panda.and the Black Bear. The cDNA of TNNC1 for the Giant Panda.and the Black Bear were transfected into E. coli, the TNNC1 fused with the N-terminally His-tagged protein gave rise to the accumulation of an expected 23.5 KDa polypeptide, which accorded with the predicted protein.