| Chinese giant salamander (A.davidianus blanchard) is a kind of native species endemic to China, called Wawayu in Chinese for its baby-like cry, which belonged to Andrias, Cryptobranchidae, Caudata of amphibians. Chinese giant salamander has nutritional value, pharmacological and health activities. Now it becomes a new type of major breeding animals in main production area. Research has demonstrated that amphibian skin secretions are rich in a variety of highly valuable active substances. Some researchers focus on plenty of natural bioactive products secreted by general skin particularly without any reported about Chinese giant salamander. A cDNA library from skin of A.davidianus not only is helpful for exploiting the biological gene resources of amphibians without damage, but also to search for some functional genes in skin of A.davidianus. Therefore, a cDNA library from skin of A.davidianus was constructured with EST sequences analysis. These genes from skin of A.davidianus were identified further to sreen for immune-related genes. All these work will be helpful for exploitation for natural bioactive products, research on immune process, growth and development, mechanism and natural resources protecting.In this study, the materials were obtained from Tian han company of Chinese giant salamander, Hanzhong, China. Skin tissue used for total RNA extraction were taken from back of A.davidianus. The skin cDNA library was constructed using Creator? SMART? cDNA Library Construction Kit (Clontech Inc.) following the instructions. Then, the titer of the cDNA library were assessed by calculating white clones on the plates and the average length of cDNA clones was determined by PCR amplification using universal M13-47 and RV-M primers. Randomly selected 400 library clones, then sequenced by TaKaRa Biotechnology (Dalian, China) using ABI 3730 automated DNA sequencer. E. coli (DH10B) DNA sequences, vector and adapter sequences were removed via Cross-match program. Analysis of nucleotide and aa sequences were performed with Vector NTI Suit 8.0 software. ESTs (>100 bp) were assembled into contiguous sequences (contigs) using Phrap program with slight modification of default settings as 40 bp minimum overlap and 99% identity. Contigs and singletons were subjected to local BLAST X and BLAST N analysis to search for similarity against the non-redundant (nr) protein and nucleotide (nt) databases. The received EST sequence of this study who matched NCBI website homology with the expected value≤1e-06 was judged to be homologous sequence. Functional classes were assigned according to Clusters of Orthologous Genes (COG) and Gene Ontology (GO) classifications.The results showed that:1. The titer of the primary cDNA library was 1.50x106 cfu, the recombination ratio of the library was about 94.8% and the average insert size was 1.0 kb.2. A total of 325 ESTs from the library were sequenced and made alignments with sequences in Gene Bank database. Moreover at least 214 genes derived from these identified clones were categorized into nine categories: immune-related genes and metabolism gene accounting for the largest distribution. The nine categories were general function prediction only, metabolism, structure, cell cycle control, secretion, protein posttranslational modification, turnover, chaperones, genomic modificating, signal transduction mechanisms, function unknown.3. There were a lot of functional genes in the library, which contained amount of mtlRNAs. Some high expression genes in the form of two classes. One was immune-related genes, just like Galectin-3, Osteoglycin (OGN, 1.5%), Ovoinhibitor precursor (1.2%), Macrophage-capping protein (1.2%), Ferritin-heavy polypeptide 1 (Fth1, 0.9%), Chorionic proteinase inhibitor (0.9%). There were also lots of metabolism genes, such as Cytochrome c oxidase subunit II (CO II, 4.3%), NADH dehydrogenase subunit 2 (1.2%), COⅢ(0.9%), Triosephosphate isomerase (0.9%), and etc.4. 7 Major immune-related genes were firstly identified from the library bescause of most distribution in A.davidianus (>0.5%). These genes were as follows: Galectin-3, Ovoinhibitor precursor, Mcp, Fth1, TribSPI, ANXA1and Tβ-4. Moreover genes related to cancer cell metabolism or microfilament migration were also found. Cystatin A1 (CSTA1), Cystatin B (CSTB) were related with meat quality. Some secreted genes were present in large amounts in expression, just like, for instance, Fth1 encodes the heavy subunit of ferritin, the major intracellular iron storage and releasing protein in prokaryotes and eukaryotes. Peptidyl-a minoacyl-L/D-isomerase precursor gene, which used in transformation of amino acids in different style. There were some other genes, Tubulin, Macrophage Migration Inhibitory Factor (MIF), and etc.5. According to the constructured cDNA library, we had isolated Arp2/3 complex subunit 5-like (Arpc5l) gene and Dynein light chain, LC8-type 2 (Dynll2) gene. Generally sequence analysis and gene annotation were analyzed by NCBI-BLAST online services. The characteristics of functional protein encoded by the ones, singnal peptide, hydrophobicity structure, transmembrance domains were analyzed by ScanSite pI/Mw software, SingalP3.0 software, Protscale software, TMHMM software respectively. Also characteristic motifs, transmembrance helices, secondary structure, predicted transmembrane structure verification were analyzed by SMART software, Tmpred software, SOPMA software, Phyre software individually. However, the spacial structure of the proteins were used Swiss-model software and checked by Swiss-Pdb Viewer software. Thus, multiple sequence alignments of AA analyzed by MEGA4.0 software and the phylogenesis tree were constructured adopting UPGMA method together with checking by Bootstrap1000 system to ascertain the accuracy. At last, we detected Arpc5l and Dynll2 mRNAs in different tissues through qRT-PCR.To analyze structure and tissue-tissue-specific expression of Arpc5l and Dynll2 genes from A.davidianus by bioinformatics analysis, Arpc5l and Dynll2 genes were isolated from skin cDNA library and revealed, which were related to motor activities, microfilament migration, or even cancer cell movement. We cloned the Arpc5l cDNA from skin cDNA libraries using our own sequence as primers. The cDNA from skin cDNA library was 699 bp in length encoded 153 AA. While Dynll2 was 682 bp in length ecoded 104 AA independently. The two genes had been revealed that they had high distribution of nucleotide"A+T", high propotion ofα-Helixs, no signal peptides, lots of alkaline residues in deduced AA sequences. and the result of the tertiary structure had revealed that Arpc5l of A.davidianus was highly homologous to Arp2/3 complex family of Bos taurus, while Dynll2 homologous to dynein family resulted from similarity to Rattus norvegicus. Searching in public database together with sequence comparisons, we assembled 10 related sequences of Arpc5l, followed by Bos taurus, Homo sapiens, Mus musculus, Xenopus laevis, Pongo abelii, Rattus norvegicus, Danio rerio, Sus scrofa, Xenopus tropicalis, Macaca fascicularis. 26 related sequences of Dynll2 were collected, concluding Ornithorhynchus anatinus, . Homo sapiens, Pongo abelii, Canis familiaris, Ailuropoda melanoleuca, Danio rerio, Mus musculus, Osmerus mordax, Esox lucius, Branchiostoma floridae, Ciona intestinalis, Epinephelus akaara, Oncorhynchus mykiss, Anoplopoma fimbria, Strongylocentrotus purpuratus, Xenopus Silurana tropicalis, Xenopus laevis, Salmo salar, Bos taurus, Nematostella vectensis, Callithrix jacchus, Branchiostoma belcheri, Sus scrofa, Rattus norvegicus, Taeniopygia guttata, and Rana catesbeiana. As a resullt, phylogenesis analysis of their genomic sequences were obtained. Moreover, The expression profile of Arpc5l in different tissues were investigated through qRT-PCR. 8 tissues of A.davidianus were kindey, bowel, muscle, liver, lung, skin, spleen and stomach. Using double-standard curves method of Arpc5l andβ-Actin mRNA, the data of gene copies had been analyzed derived from their own standard curve. The results of qRT-PCR indicated the plentiful presence of Arpc5l mRNA in kindey, bowel and muscle with significant difference (P<0.01). Dynll2 mRNA in 6 tissues of A. davidianus were also detected, followed by bowel, muscle, liver, lung, skin and stomach. The results of qRT-PCR, data analyzed by 2-△△Ct method, indicated the plentiful presence of Dynll2 mRNA in muscle rather than other 5 tissues. |
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