| As one of China’s four major farming shellfish, the razor clam Sinonovacula constricta possesses the important economic status in aquaculture. The study of the gene relating growth and development contributes to the selective breeding of S.constricta and improving the economic value. In this research, the development transcriptome library of S.constricta was contructed and three insulin-like peptide genes(ILP) were found. ILP is one important member of the insulin-like superfamily, which plays an essential role in the growth and metabolism regulation of the organism.1. Characteristics analysis of the development transcriptome libraryUsing a new generation of Illumina HiSeq 2000 high-throughput sequencing technologies, the development transcriptome library of S.constricta was built for the first time, including post-fertilization embryos, trochophores, veliger larvae, umbo larvae, creeping larvae and juvenile clams. A toltal of 249,795 reads were sequenced. Gene length was from 201 bp to 36,941 bp, with an average of 585 bp. And the longest annotation gene was Nesprin-1(11087 amino acids). Through the online program BLASTx, the splicing sequences were compared with gene sequences has publiced. After comparing, 21920(8.78%), 8837(3.54%), 14036(5.62%), 15978(6.4%) genes were annoted in the NCBI Nr, COG, Swiss Prot and KEGG program respectively. Through program GO, 9205 genes can be classified as three main functional domains: biochemical processes, cellular component and molecular function. Some genes were identified connecting with the metamorphosis of S.constricta. Primers were designed according to the ORF of these genes for qPCR. The results showed that they play a very important role in the metamorphosis of S.constricta. The development transcriptome of S.consricta enriched the genetic information of S.consricta, and had important reference value for the further study of molecular mechanisms of S.consricta larvae growth and development.2. Cloning and genetic structure analysis of insulin-like peptide genes.In this study, three members of insulin-like superfamily, ILP1, ILP2 and ILP3 genes were cloned, and the full length cDNA sequence of these three genes were obtained by RACE technology, containing 1339 bp, 1017 bp and 2415 bp respectively. The length of ORF of three ILP1 genes was 525 bp, 537 bp and 438 bp, encoding 174, 178 and 145 amino acids respectively. Gene sequence analysis showed that these three genes have signal peptide and conservative domain, which was divided into three peptides, B-chain, A-chain and C-chain. The six cysteines formed three disulfide bonds, two inter-chains disulphide bridges between B-chain and A-chain, one intra-chain disulphide bridges within A-chain, the three disulfide bond determined the protein structure and function of the ILP gene.3. Expression analysis of insulin-like peptide genes.The expression profile of ILPs were studied, including seven adult health organizations(mantle, foot, siphon, gill, digestive gland, and gonads, and hemocytes) and six larvae period(post-fertilization embryos, trochophores, veliger larvae, umbo larvae, creeping larvae, juvenile clams). The results showed that ILP1 had a higher expression in the gills and foot, while ILP2 expressed higher in siphon and foot. In different developmental period, ILP1 had highest expression in the juvenile clams. Both ILP2 and ILP3 had a higher expression in the development of the early and middle period.4. The effect of insulin-like peptide genes on S.constricta gonad development process.The gonad tissue was collected during the developmental process and observed the development of gonad through frozen section. There were four stages of gonadal development, including proliferation stage, growth stage, maturity stage and diffusion stage. According to the expression profile of ILP2 and ILP1 in each developmental stage, the expression of the two genes was higher in the early stage of gonadal development in male and female individuals.5. The preliminary detection of ILP receptor and cell proliferation analysisThrough the comparison of amino acid sequences, ILPR of S.constricta had higher similarity with insulin receptor of human than IGF1 receptor and IGF2 receptor. Three genes were connected to the pEGFP-N1 vector, and transfected into human 293 T cells, over-expressed this three genes. After transfection, the expression changes of insulin receptor, IGF1 receptor and IGF2 receptor in 293 T cells were detected. The results showed that insulin receptor gene expression was up-regulated after transfection and the expression of IGF1 and IGF2 receptors genes had no significant changes. Numbers of cells by flow cytometry were found that in the N1 vector plasmid control group, the cell growth rate was 2.56. In ILP1, ILP 2 and ILP 3 transfection group, the cell growth rate was 5.73, 3.15 and 8.79 from 0 to 48 h, respectively. |
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