Bioinformatics Analysis And Expression Research Of Tβ-4Gene And Hb-αl Gene From Chinese Giant Salamander

Chinese Giant salamander (Andrias davidianus) is commonly known asWawayu in Chinese for its baby-like crying,which belonged toAndrias,Cryptobranchidae,Caudata of amphibians. It is a kind of native speciesendemicnto China,and also its evolutionarily is quite primitive,because this specie isjust between aquatic to terrestrial transition. Chinese Giant salamander are mainly inthe the creek canyon of the upper reaches of the Yangtze River, Pearl River and HanRiver.Whatsmore, Chinese Giant salamander has high water quality requirements forits survival, just like the clear water and low temperature. In recent years, due to theenvironmental pollution and human hunting, Chinese Giant salamander’s natureresources were seriously destroyed. Because Chinese Giant salamander is one specieof China’s endangered treasure animal protection, so the current research mainlyfocused on its conservation genetics,which provides important meaning to germplasmresource management and artificial proliferation, While the relative research of skinactive agent of Chinese Giant salamander is quite less than that.In this case, thymosin β-4cDNA sequence and Hemoglobin-α1cDNA sequencewere isolated and identified from skin cDNA library of Chinese giant salamander. Wedid the bioinformatics analysis of the full-length cDNA sequence by a lot ofbioinformatics software,such as the sequence similarity analysis and gene annotationof thymosin β-4and Hemoglobin-α1were analysed by BLASTN and BLASTX inNational Center for Biotechnology Information（NCBI）;The properties of proteinswere finished by ScanSite pI/Mw software; SingalP4.0web server could help to findout whether there was signal peptide exsisted in sequence; Protscale software mayanalysis hydrophobic structure, TMHMM predicted transmembrane structure andSMART software wered usedfor protein features motif detection; Tmpred softwarepredicted transmembrane helix, SPOMA predicted the secondary structure, Phyre didverification and Swiss-model did three structure prediction. We tested theHemoglobin-α1gene and thymosin β-4gene’s difference in expression quantity in the intestinal, muscle, skin, stomach, kidney, lung, liver of Chinese Giant salamander byusing qRT-PCR method. The results showed that:(1) In this case, Hemoglobin-α1cDNA sequence was isolated and identifiedfrom skin cDNA library of Chinese giant salamander.The Hemoglobin-α1gene was594bp in the full length with the ORF432bp. Besides,144kinds of amino acids wereencoded by Hemoglobin-α1gene, and the molecular weight was16048.3u in general,isoelectric point was6.44. In addition, the structure analysis of Hemoglobin-α1showed that the composition of amino acids were acidic amino acids11.9%, neutralamino acids69.9%and basic amino acid18.2%. Structural analysis indicated thatthere was no signal peptide existed in this protein but there was an outwardtransmembrane helical region from100thto122ndamino acids and also a inwardtransmembrane helical region from98thto122ndamino acids, whose secondarystructure was mainly α-helix.The three-level structure of Hemoglobin-α1proteinindicated that the evolutional relationship was that Andrias davidianus washomologous to land animals, with Homo sapiens and Macaca mulatta homology ofHemoglobin-α1was nearly63.0%, secondly were Sus scrofa, Mus musculus andXenopus laevis, homology is about59%, and Rana catisbianna took the lowesthomology (46.7%). Moreover, we were able to analyse the expression ofHemoglobin-α1gene mRNA in7tissues for Andrias davidianus by the data ofqRT-PCR, it was more expressed in muscle than the other six issues and expressedleast in stomach.(2) In this case, thymosin β-4cDNA sequence was isolated and identifiedfrom skin cDNA library of Chinese giant salamander. The thymosin β-4gene was699bp in the full length with the ORF204bp (19bp-223bp),5’-UTR87bp，3’-UTR474bp. Besides,68kinds of amino acids were encoded by thymosin β-4gene, inwhich43amino acids were mature peptide and the molecular weight was7649.7u ingeneral, isoelectric point was5.81. In addition, the structure analysis of thymosin β-4showed that there was a “THY” specific motif, but no transmembrane helix andtransmembrane domain existed. The three-level structure of thymosin β-4proteinindicated the evolutional relationship was that Andrias davidianus was homologous toland animals, with Xenopus and Lymnaea homology of thymosin β-4was over80%,and rainbow took the lowest homology (62.2%). Moreover, we were able to analysethe expression of thymosin β-4gene mRNA in7tissues for Andrias davidianus by thedata of qRT-PCR, it was more expressed in lung than the other six issues. In addition, in this case, the construction of prokaryotic fusion expression vectorof mature peptide (pET-32a-Tβ4) of Chinese Giant Salamander thymosinβ-4was byusing artificial synthesis,and expressed in E. Coli BL21. We transformed7rarecodons amino acids such as Arg, Gly, Leu of the giant salamander thymosinβ-4geneinto Escherichia coli favored codon. Expressed the synthetic thymosinβ-4maturepeptide, HisTag and pET32a vector of thioredoxin fusion of Chinese Giantsalamander. Protein electrophoresis showed that the fusion protein containing thepresent mature peptide was efficiently expressed after1mmol/ml IPTG induction at30℃for4h at proximity to26kDa, and the protein molecular weight was nearly5kDa in the removal of fusion expression of thioredoxin redox protein and HisTag,which was the same to the expected target protein size consistent. The result reflectedthat the thymosin β-4gene’s prokaryotic expression vector was successful constructed,which would be the basis for the further study on the genes of Andrias davidianusphysiological regulation mechanism.