The Function Study Of Cathepsin B Gene In Ovary Development Of Marsupenaeus Japonicus

Cathepsin B(CB)is a proteinase of the cathepsin superfamily,which also acts as an endopeptidase and dipeptidyl carboxypeptidase at the same time.CB widely exists in tissues of aquatic animals,and it expresses differently in different species.High expression of CB in the ovary indicates that it plays an important role in ovarian development,egg formation and embryonic development.Its high expression of immune organs undering conditions of stress by viruses or bacteria,such as in the blood,implys that it may be involved in the process of immune challenge.Therefore,CB plays a key role both in vertebrates and invertebrates.Through the differential proteomics in Marsupenaeus japonicas,we have got one of the ovary specific proteins and cloned full length c DNA of CB of Mjaponicas(Mj CB).Based on this,the genome DNA(g DNA)and 5'regulatory region were obtained by genome walking and conventional PCR.Furthermore,throughing the construction of plasmid and induced expression,we also get the polyclonal specific antibody.By RNA interference(RNAi)experiments of adult,the expression of genes related to ovarian development were analyzed in ovarian tissue.In addition,through the experiment of unilateral eyestalk removal,we got the expression of genes related to ovarian tissue.Conclusions are as follows:(1)Mj CB's g DNA was cloned for the first time by means of genome walking method combined with convertional PCR.The full-length is 3122 bp,and g DNA consists of four exons and three introns.The first exon is 20 bp and the first intron is 1 440 bp.The intron shear follows the typical “GT-AG” rule,and the translation initiation codon located in the second exon.(2)Using bioinformatics software to analysis the 5'regulatory region,we predicted one core promoter regions,one transcriptional start site and one Cp G island.There are 29 transcription factor binding sites,such as Oct-1,F-kappaB,SP1,AP-1,GATA-1 and so on.(3)Using double label method(His/GST)to construct the prokaryotic expression recombinant plasmid p GEX-4T-2-Mj CB successfully,we got the soluble protein of Mj CB after renaturation.The polyconal specific antibody which can be used in the western blot detection was got after purification.(4)Mj CB's expression of M.japonicas in different tissues on ovarian development of phase II stage was studied by Quantitive Real-time PCR(q PCR).The results showed that Mj CB's expression in the ovarian tissue was significantly higher than that in other tissues.(5)The recombination of about 400~650 bp of Double Stranded RNA(ds RNA)in vitro was used in the RNAi experiment.And the results showed that 12 h after interference was the optimal phase by q PCR.(6)In the study on genes related to interferce experiment,we adopt two control groups injection of ds EGFP and injection of physiological saline group.This can ensure the reliability of the experimental data.When CB was interferenced,Vitellogenin(Vg)gene expression was significantly down regulated(P<0.05)and Cathepsin C(CC)gene was significantly up-regulated(P<0.05),however,the Cathepsin L(CL)gene was not affected(P>0.05).(7)In unilateral eyestalk ablation group,CB gene was significantly down regulated(P<0.05),but Vg,CC,CL gene's expression was not significant compared to the control group(P>0.05).