Cloning,Expression And Functional Analysis Of CDK2 And P53 From Black Tiger Shrimp (Penaeus Monodon)

Farming of the black tiger shrimp(Penaeus monodon) has considerable economic importance in the world. In the eyestalk of P. monodon have ovarian suppression hormones, so unilateral eyestalk ablation can promote ovarian maturation in females. Although unilateral eyestalk ablation is practically used to induce ovarian maturation of P. monodon, this technique usual y leads to stress and the death of spawners. To avoid the use of eyestalk ablation, understanding molecular mechanisms controlling the development and maturation of ovaries/oocytes is essential for finding an alternative approach to trigger the reproductive maturation of P. monodon without the negative effects from eyestalk ablation. In this study, CDK2 and p53 cDNA sequence fragme nts were obtained from the hepatopancreas transcriptome data of black tiger shrimp, on this basis the ful- length cDNA sequences of PmCDK2 and Pmp53 were cloned by sequencing and SMART-RACE, and then carried out bioinformatic analysis respectively. Multiple sequence alignment and phylogenetic analysis of PmCDK2 and Pmp53 were performed with previously published amino acid sequences of other species, respectively. The temporal expression of PmCDK2 and Pmp53 in different tissues and differe nt developmental stages of ovary were investigated by qRT-PCR, respectively. The technology of RNAi was used to determine the effects of p53-dsRNA on Pmp53 and PmCDK2 gene expression.We performed preliminary study of the interplay between Pmcyclin E and PmCDK2 in vitro. The main results are as follows:Cyclin-dependent kinases(cdks) are key regulators of the cell cycle. It and cyclin(Cyclins), cyclin-dependent kinase inhibitor(CKIs) and other components compose the cell cycle regulatory network systems, regulation of cell cycle progression together. Each cdk binds with its corresponding cyclin forming a complex that functions as a catalytic subunit to regulate a certain phase of the cell cycle. In the study, the ful- le ngt h complementary DNA(cDNA) sequence of CDK2 from black tiger shrimps P. monodon(PmCDK2) was obtained by sequencing and rapid amplification of cDNA ends(RACE). The cDNA of PmCDK2 was 1679 bp, including a 258 bp 5’-terminal un-translated region(UTR), a 500 bp 3’ UTR with a poly(A) tail, and an open reading frame(ORF) of 921 bp encoding a polypeptide of 306 amino acids with a predicted molecular weight of 34.91 kD and predicted pI of 7.02. Blast and phylogenetic analyses suggest that PmCDK2 is a new member of the shrimp CDKs family. PmCDK2 mRNA expression was detected in seven tissues by quantitative real-time PCR(qRT-PCR). Expression was the highest in the testis and moderate in the ovary, lymph, muscle et al. While at different stages of ovary development, PmCDK2 expressed highest at stage III, indicating that PmCDK2 might play an important role in ovarian development for black tiger shrimp. Constructed CDK2-pET21 a recombinant plasmid and then transformed into E. coli, carry out prokaryotic expression experiment, successful get a fusion protein containing six His tag. Western Blot to detect the fusion protein. The cyclin E/CDK2 complex plays a key role in the G1/S transition of the cell cycle. However, the knowledge of the interplay between cyclin E and CDK2 is still limited. In this study, recombinant proteins of the Pmcyclin E and PmCDK2 were expressed in E. coli, and purified by Ni-chelating affinit y chromatography. Pull-down experiment was performed to detect the interplay between Pmcyclin E and PmCDK2. Overall, this study will be useful to study the functio na l mechanisms of the cyclin E/CDK2 complex in shrimp, which would further enrich the knowledge of cell cycle regulation in invertebrate.This is the first report on the cloning and expression pattern analysis of the CDK2 in P. monodon. Our results provide insight into the function of PmCDK2 in the regulation of ovarian development in the black tiger shrimp.The tumor suppressor p53 is a sequence-specific transcription factor, whose target genes can regulate genomic stability, the cellular response to DNA damage and cell-cycle progression. In the present study, the full-length cDNA sequence of p53 gene from P. monodon(Pmp53) was cloned by the technology of RACE. The cDNA of Pmp53 was 2,239 bp, including an open reading frame(ORF) of 1,353 bp that encoding a peptide of 450 amino acids with calculated molecular weight of 50.62 kDa, a 5’UTR of 122 bp, and a 3’UTR of 764 bp. Blast and phylogenetic analysis discovered that the predicted amino acid sequence of Pmp53 shares high homology and the close phylogenetic relations hip with that in Litopenaeus vannamei and Marsupenaeus japonicus indicate quite similar regulation roles of p53 in these species. The temporal expression of Pmp53 in differe nt tissues(ovary, heart, intestine, brain, muscles, stomach and gills) and differe nt developmental stages of ovary was investigated by qRT-PCR. The Pmp53 gene were expressed in all the examined tissues, with relatively high levels observed in the brain and heart, moderate levels found in the gil, ovary, and intestines, and low levels detected in the muscle and stomach. The lowest expression level of Pmp53 was observed in the stomach, while the highest expression level was detected in the brain. During the ovar ian development stages, the expression level of Pmp53 reached the peak at Stage III, about 5-fold greater than that in other stages. RNA interference(RNAi) and serotonin(5-hydroxytryptamine, 5-HT) injection experiments were conducted to study the express ion profile of Pmp53 and PmCDK2. Knocked down of Pmp53 by dsRNA-p53 was sequencespecific and successful.The relative expression level of Pmp53 mRNA significa nt ly upregulated, and the relative expression level of PmCDK2 mRNA notably up-regulat io n in ovary and hepatopancreas post injection of p53-dsRNA. The expression level of Pmp53 and PmCDK2 in ovary of P. monodon were both significantly increased after injection of 5-HT, but the former is much lower than the latter. In this study, to inspect the protein function of Pmp53, the recombinant protein of Pmp53 was constructed in vitro and analyzed by SDS-PAGE and Western-Blot. These results indicate that Pmp53 may be involved in the regulation of ovarian development of P. monodon.In the study, we obtained the characterization of the full- length cDNA of Pmp53 and PmCDK2 gene; the temporal expression of PmCDK2 and Pmp53 in different tissues and different developmental stages of ovary were investigated by qRT-PCR; RNAi experiments revealed that Pmp53 down-regulate the expression level of PmCDK2 and ensure a smooth process of the cell cycle. Therefore, PmCDK2 and Pmp53 may influe nce the cell cycle, and then affecting the ovarian development of P. monodon. These research results provide a theoretical basis for further study the mechanism of ovarian development of P. monodon.