CDNA,Genomic Sequence,Overexpression And Related Research Of BANF1 Gene Of The Giant Panda And The Black Bear

The giant panda(A.melanoleuca) known as a “living fossil” is a rare species currently found only in China. Its distribution range includes the northwest of Sichuan, southwest of Shanxi, eastern Tibet and southern Gansu. Its main food is bamboo, also love to eat honey. Due to the low reproductive rate and Small population, It has been listed as endangered primate national level. The black bear(Ursus thibetanus mupinensis) names also include Moon Bear, Tibetan bears, It is belongs to omnivorous animals and the main feed plants,berries and fungi. It is lives mainly in southern Asia, due to large quantities hunted, It is has been listed as two national key protected wild animals and inclusion of China Red Data Book of Endangered Animals. Both species have a very important research value.Primers were designed as the related m RNA sequences of five mammalians, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Pan troglodytes. Extraction of total RNA and genomic DNA from the skeletal muscle of the Giant Panda and the black bear. We used Touchdown PCR and RT-PCR technique to amplify and cloning the genomic sequence and c DNA o f BANF1 gene both of the Giant Panda and the Black Bear. We obtained sequences were sequenced, and then analyzed the sequence characteristics of c DNA and compared it with others reported. We constructed the recombinant expression vector which involved BANF1-c DNA, then over-expression it in E.coli- BL21 and inducing expression of the protein what we want. Which accumulate schemes and experiences pushing research progress at the molecular level.Both sequences were analyzed preliminarily, The c DNA of the BANF1 cloned from the Giant Panda and the Black Bear is 297 bp in size, containing an open reading frame of 270 bp encoding 89 amino acids. The length of the genomic sequence from Giant Panda is 521 bp, from Black Bear is 536 bp, which were found both to possess 2 exons. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to some mammalian species studied.As determined by BLAST analysis, the nucleotide sequence BANF1 cloned from the Giant Panda shares a high homology with those of Ursus thibetanus mupinensis, Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Pan troglodytes of 98.15%,96.6%,93.3%,93.03%,96.6 % and 96.6%,respectively. The homologies for amino acid sequences is 96.63%,93.7%,92.6%,91.5%,91.1% and 93.7%, respectively. the nucleotide sequence BANF1 cloned from the Black Bear shares a high homology with those of Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Pan troglodytes of 95.19%,94.10%,93.33%,92.96 and 95.56%, respectively. The homologies for amino acid sequences is 100%,96.63%,96.63%,100% and 100%, respectively. Topology prediction showed there is one Protein kinase C phosphorylation site, one Tyrosine kinase phosphorylation site, one Amidation site and one N- myristoylation site in the BANF1 protein of the Giant Panda and the Black Bear; in addition, there is one Casein kinase II phosphorylation site in the BANF1 protein of the Giant Panda. The BANF1 gene can be readily expressed in E. coli BL21, Results showed that the protein BANF1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 14 k Da polypeptide that formed inclusion bodies. The expression products obtained could be used to purify the proteins and study their function further.