Molecular Cloning And MRNA Expression Of Differential Expressed Genes During Ovarian Development Of White Prawn(Litopenaeus Vannamei)

The problem of shrimp ovarian maturation under artificial conditions has been a inhibition of breeding production.Although the method of eyestalk-ablation can promote maturation and reproductiom of the broodstock but it is destruction. So the focus and important issue of shrimp reproductive and developmental biology is to explore new regulation mechanism of vitellogenesis. In this study, we use Litopenaeus vannamei as the research object and select the nuclear autoantigen sperm protein gene（NASP） and the polehole-like protein（PHL）gene as research gene by suppression subtractive hybridization（SSH）. The study will be foundation for the research of mechanism of ovarian development of L. vannamei.In the study,we cloned full-length cDNA sequences of two genes by rapid amplification of cDNA ends（RACE）. Two genes mRNA expression levels were investigated in ovary and hepatopancreas of L.vannamei at different developmental stages by real-time fluorescent quantitative PCR.The results are as follows: 1) The full length cDNA of nuclear autoantigenic sperm protein（ NASP） consists of 2,258 bp with 92-bp 5’-untranslated region（UTR）,174-bp 3’-UTR and 2,019-bp Open Reading Frame（ORF）, which encoded 673 amino acids and the isoelectric point（PI） was 4.46,the predicted molecular mass was 74.18 k D. The presumed protein had two conserved regions: SHNi-TPR,TPR₂ and 4 Glycosylation sites.The sequence has been submitted to GenBank, and the accession number is KT274811.Sequence alignment analysis by NCBI showed that the NASP gene between L.vannamei and P.monodon（Gen Bank number: FJ040859.1） has the highest similarity of 90%. Furthermore, we constructed the phylogenetic tree based on NASP sequences to evaluate the evolutional relationship between L.vannamei and other species. The analysis revealed that the NASP in L.vannamei was in the same branch as that of P.monodon. Relative NASP mRNA expression levels were investigated in ovary and hepatopancreas of L.vannamei at different developmental stages by real-time fluorescent quantitative PCR. The results showed that NASP mRNA was expressed at all the stages, and the expression in the ovary is higher than that of the hepatopancreas, and which is highest at second stage, while at third stage is lowest. Compared with ovary, the expression level of NASP in the hepatopancreas is ignorable. Expression was no significant difference between every period. The analysis showed that NASP function was associated with cell division activity.2)The full length cDNA of polehole-like protein（PHL） consists of 6,343 bp with 117-bp 5’-untranslated region（UTR）,61-bp 3’-UTR and 6,165-bp Open Reading Frame（ORF）,which encoded 2,054 amino acids and the isoelectric point（PI） was 4.84,the predicted molecular mass was 224.46 KD. The presumed protein had two conserved regions: PbH1, EPTP and 12 Glycosylation sites,23 transmembrane.The sequence has been submitted to Gen Bank, and the accession number is KU981017.Sequence alignment analysis by BLASTP showed that the PHL protein sequence between L.vannameian and P. monodon（GenBank number: ACV60547.1） has the highest similarity of 75%. Relative PHL m RNA expression levels were investigated in ovary and hepatopancreas of L.vannamei at different developmental stages by real-time fluorescent quantitative PCR. The results showed that the expression in the ovary is higher than that of the hepatopancreas, and which is significantly high at first stage while at fifth stage is lowest. The expression recovered slightly at sixth stage. In addition to a lower amount of expression of the sixth, there was no significant difference between each other stages of hepatopancreas. The results will provide a basic for further study of L. vannamei ovarian development.