Molecular Cloning And Expression Analysis Of Some Cell Cyclin Genes From Black Tiger Shrimp (Penaeus Monodon)

To the development of prawn breeding industry, the technology of parent shrimpartificial breeding is an important limiting factor. Because female shrimp can’t reachovarian maturity themselves. And dur to the method of eyestalk-ablation is destructiveand the wild-caught broodstock is too cost for the breeding, it is imperative for us toexplore new prawn breeding method[1,2]. In the present study, we clone and analysePmcyclinH, PmcyclinE,etc. The study will be foundation for the research of mechanismof ovarian development.PmcyclinH was identified from black tiger shrimp Penaeus monodon by ESTanalysis and RACE approaches. The full length cDNA of PmcyclinH is of1280bp,including a5’-terminal un-translated region (5’UTR) of63bp, a3’UTR of218bp witha poly (A) tail, and an open reading frame (ORF) of999bp encoding a polypeptide of332amino acids with a predicted molecular weight of39kDa, predicted pI of6.39.Blast and phylogenetic analysis together supported that PmcyclinH was a new memberof shrimp cyclin H. The mRNA expression of PmcyclinH in eighteen tissues wasexamined by real-time PCR, and mRNA transcript of PmcyclinH was predominantlydetectable in the tissue of ovary, to a less degree in the tissues of intestine and stomach,liver, heart and brain, but almost not detected in the muscle. The temporal expression ofPmcyclin H in different developmental stages of ovary was investigated by real-timePCR. During the five stages of ovary development, two peaks expression of PmcyclinHwas detected in stage II and stage Ⅵ. All these results indicated that Pmcyclin H mightbe involved in the regulation of cell cycle and ovary development of P. monodonThe full-length cDNA sequence of cell cyclin E from black tiger shrimps Penae usmonodon (denoted as PmcyclinE) was obtained by high throughput transcriptomesequencing and RACE approaches. The cDNA of PmcyclinE was of1706bp, includinga5’-terminal un-translated region(5’UTR) of132bp, a3’UTR of311bp witha poly(A)tail, and an open reading frame (ORF) of1263bp encoding a polypeptide of420amino acids with a predicted molecular weight of47.9kDa, predicted pI of5.85. Blast andphylogenetic analysis together supported that Pmcyclin E was a new member of shrimpcell cyclin E. The mRNA expression of PmcyclinE in nine tissues was examined byreal-time PCR, and mRNA transcript of PmcyclinE was predominantly detectable in thetissues of ovary and eyestalk, to a less degree in the tissues of intestine, brain andmuscle, but almost not detected in the stomach and hepatopancreas. Pmcyclin E ofovary was significantly repressed towards injection of MIH, and was induced aftereyestalk depletion, suggesting that the gene plays an important role in black tigershrimp’s ovary development, These results provides a basci information for furtherexploring the gene function in shrimp’s ovarydevelopment regulation.The purified recombinant protein of PmcyclinB was obtained by Prokaryoticexpression method. Subsequently, the anti-PmcyclinB antibodies was prepared (denotedas anti-cycB) and the distribution of PmcyclinB in ovary was analysed byimmunohistochemistry (IHC) method, found that the expression of PmcyclinB proteinof the control group significantly lower than that of the experimental group.