Molecular Cloning,Recombinant Expression And Transcriptional Regulation Analysis Of VIH Gene In Scylla Paramamosain

Vitellogenesis-inhibiting hormone?VIH?is an important neuropeptide which belongs to the member of crustacean hyperglycemic hormone?CHH?family.It is synthesized and secreted from the X-organ/sinus gland complex in the eyestalks,which plays a crucial role in the regulation of vitellogenesis and reproduction in decapods.In this study,the partial sequence of VIH gene was identified from our previous study.The full length cDNA and gDNA,and the5'upstream regulatory sequence of VIH were cloned by genome walker,Tail-PCR and conventional PCR.Meanwhile,the recombinant expression in vitro,cell culture,transient transfection and dual-luciferase reporter assays were also used to analyze the structure characteristics and transcriptional regulation of VIH at transcription and translation levels.The main results of this study were as follows:1)The full length cDNA of SpVIH is 634 bp,which was cloned by genome walker.The open reading frame?ORF?is 375 bp encoding 125 amino acid residues,including signal peptide?1-22 aa?and mature peptide.Six conserved cysteines?Cys?residues and one special Gly¹²residue were identified from in the deduced mature peptide.Sequence comparison and phylogenetic analysis showed that SpVIH belongs to the type II peptide of CHH family.The analysis of three-dimensional space structure indicated that SpVIH consists of five?-helices and several irregular crimps.2)The full length gDNA of SpVIH was 790 bp containing two exons and one intron which was cloned according to the sequence of the cDNA by conventional PCR.The exon and intron were both located in the ORF,and the intron was between Arg?AGG?and Arg?AGG?.However,the splice donor and acceptor sequence of the intron did not follow the typical“GT-AG”or“AT-AC”rules.3)The mature peptide of SpVIH was recombinant expressed in vitro by prokaryotic expression.The SDS-PAGE analysis showed that there are two molecular weight of about 29kDa and 31 kDa,respectively.Utilizing the high resolution LC-MS/MS system,we identified that COV%?fraction of coverage?of the two protein were 57.8%and 61.8%,respectively.Therefore,the two protein were deduced as objective proteins.Meanwhile,the mature protein of about 29 kDa was purified by Ni column and the antibody was prepared in rabbit.Furthermore,western blot analysis showed that the antibody could be combined with both of the two objective proteins.4)The 5'upstream regulatory sequence of SpVIH gene was cloned by genome walker and Tail-PCR.From the translation start?ATG?and the predicted transcription start?A?,the length obtained were 3070 bp and 2398 bp,respectively.The CpG islands distribution analysis showed that there is no CpG island.The core transcriptional start region were located within-203 bp-+213 bp through bioinformatics technology and the analysis of transcriptional activity of promoters.The predicted transcription start site?A?was among the region of-203 bp-+213bp,and the upstream of-28 bp was TATA box.When the promoter sequence of SpVIH-1 was recombined into pEGFP-1 vector,it was able to drive the expression of EGFP protein in HEK293FT cells.5)The seven deletion fragments?pSpVIH-1-7?were constructed to the luciferase report gene vector pGL3-Basic,and then transfected into HEK293FT cells.The different relative fluorescence activity showed that fragment of p SpVIH-4 has higher activity,but reduced significantly in pSpVIH-5,and then increased significantly in pSpVIH-6.Results suggested that there are both positive and negative transcription factors binding sites presented between pSpVIH-4 and p SpVIH-6.In order to identify the crucial transcription factors binding site in this region,the site-directed mutagenesis of Sox9 binding site of pSpVIH-4 was created.The results demonstrated that the transcriptional activity of pSpVIH-4?decreased highly significantly?p<0.05?.Thus,it is reasonable to decuce that the Sox9 may be the important positive transcription factors which regulates the expression of SpVIH.