Cloning And Expression Of FABP4、FABP5 And RPS9 Of The Giant Panda

The Giant Panda (Ailuropoda melanoleuca), is known as "china national tresure" in China. It is a typical wild animal only surving in China and one of the most valuable species in the world. It is also the world’s most endangered species,It is the world’s wildlife conservation flagship animal known as the "living fossil",.At the same time, the giant panda because of low fertility rate and its survival and conservation status are concerned by our country and the world community. No reported at home and abroad is closely related to the giant panda health FABP4、FABP5 and RPS9 gene cloning and expression.This paper was conducted using RT-PCR technique and Touch-down technology to amplify the cDNA sequence and the genomic sequence of FABP4 gene FABP5 gene and RPS9 gene from the total RNAs and genomic DNA extracted from the skeleton muscle of the Giant Panda repectively and then we analyzed the sequence characteristics and compared it with those of human and other mammalian species reported. We constructed the recombinant expression vector (pET28a) containing FABP4、FABP5and RPS9 cDNA and transformed it into Escherichia coli BL21 which was then induced to express the protein of interest.The result indicated that the cDNA fragment of the FABP4 cloned from the Giant Panda is 470 bp in size, containing an open reading frame of 399 bp, and deduced protein was composed of 132 amino acids with an estimated molecular weight of 14.6969-kDa and PI of 8.51. The length of the genomic sequence is 3975bp, which was found to possess four exons and three introns.And the cDNA fragment of the FABP5 cloned from the Giant Panda is 495 bp in size, containing an open reading frame of 408 bp, and deduced protein was composed of 135 amino acids with an estimated molecular weight of 15.1333-kDa and PI of 6.60. The length of the genomic sequence is 3962bp, which was found to possess four exons and three introns too.And the cDNA fragment of the RPS9 cloned from the Giant Panda is 637 bp in size, containing an open reading frame of 585 bp, and deduced protein was composed of 194 amino acids with an estimated molecular weight of 22.60-kDa and PI of 11.31. The length of the genomic sequence is 6193bp,Through research we found that the giant panda of recombinant protein can efficiently express in prokaryotes, expressed in 4 hours can reach the peak. Topology prediction shows there are seven Protein kinase C phosphorylation sites, two Casein kinase Ⅱ phosphorylation sites, two N-myristoylation sites in the protein and one Cytosolic fatty-acid binding proteins signature in protein FABP4.and there are three N-glycosylation sites three Protein kinase C phosphorylation sites, one Casein kinase II phosphorylation sites, one N-myristoylation site, one Amidation site and one Cytosolic fatty-acid binding proteins signature in protein FABP5. Through comparative analysis we found that the genetic structure of different species and protein loci exist difference, these differences needs further research.Topology prediction showd that in RPS9 protin of giant panda there are:three protein kinas C phosphorylation site,two casein kinase II phosphorylation site,five N-myristoylation site and one ribosomal protein S4 signature.Alignment analysis of FABP4 and FABP5 of the Giant Panda and those of Homo sapiens, Rattus norvegicusjios Taurus, Ovis.ariea indicated that both the nucleotide sequence and the deduced amino acid sequence are highly conserved. As determined by BLAST analysis, the nucleotide sequence FABP4 cloned from the Giant Panda shares a high similarity with those animal of 93.5%、83%、90.5% and 86% respectively; the similarities for amino acid sequences are 95.5%、87.9%、87.1.5% and 84.1% respectively compared with the four species..And FABP5 shares a high similarity with those of H.sapiens, Rattus norvegicus, Bos Taurus,O.ariea of 93.5%、83%、90.5% and 86% respectively; the similarities for amino acid sequences are 95.5%.87.9%.87.1.5% and 84.1% respectively. And RPS9 shares a high similarity with those of H.sapiens, M. musculus Rattus norvegicus, Bos Taurus, of 92.65%、88.03%、87.01% 和 97.11% respectively; the similarities for amino acid sequences are 99.48%.99.48%.99.48% and99.48% respectively.This paper successfully study on the giant panda, the giant panda of FABP4、FABP5 and RPS9 gene cloning, enrich the genetic resources of the giant panda;Overexpress product for further protein function research provides research material and related basic data. The differences of FABP4, FABP5 and RPS9 genes and their coding proteins can be used as the effective molecular markers for the conservation genetics, species evolution and species identification of rare animals..