Reseach Of Some Hormones And Genes Being Correlation With Ovary Development From Penaeus Monodon

In order to surmount the trouble that the mature phase of the Penaeus monodonfemale gonad are different under nature environment, it is a important and hot spot tofind a new, safe and efficient method for forced ripening via researching breedingmechanism. In this assay, one functional gene correlated with the regulation of ovarydevelopment M-phase phosphoprotein6(MPP6) was cloned. Its expressionmechanism is preliminarily explored, along with two other genes from cyclinB signalpath. At the same time, we detected the concentration of four hormones in hemolymphin the stages of ovary development. The study could offer some basic reference forfurther research about ovary development.The techniques of paraffin section and haematoxylin-eosin staining were used toclassifying and identifying periods of ovary development in Penaeus monodon, togetherwith techniques of anatomy, cytology and histology. We used ovary developmentexternal characteristics and oocytes characteristics as our reasons of judgment. a.Biopsy shows that the majority cells are the oogoniums in the stageⅠovary; b. Biopsyshows that most of the cells are chromatin nucleolus oocytes and perinucleolus oocytesin the stageⅡovary; c. Biopsy shows there are a small number of yolky oocytes in thestage Ⅲ ovary; d. Biopsy shows that the largest number of cells are yolky oocytes in thestage Ⅳ ovary; e. Biopsy shows that the oocytes bestrew with cortical rod oocytes onthe edge in the stage Ⅴ ovary; f. Biopsy shows that the most of the cells are chromatinnucleolus oocytes and perinucleolus oocytes and some are cortical rod oocytes in thestage Ⅵ ovary.The techniques of Rapid Amplification of cDNA Ends (RACE) were used to clonethe Penaeus monodon M-phase phosphoprotein6gene (PmMPP6). The full lengthcDNA of PmMPP6contained a5’untranslated region (UTR) of68bp, an ORF of450bp encoding a polypeptide of149amino acids and a3’UTR of172bp. The estimatedmolecular mass was17.01kDa and the theory isoelectric point was4.89. Sequencealignment analysis showed that the PmMPP6and Culex quinquefasciatus shared a highsimilarity and homology. The techniques of chemiluminesence immunoassay（CLIA）were used to detect theconcentration and variation tendency of luteinizing hormone (LH), follicle-stimulatinghormone (FSH), progesterone (P) and estradiol (E2) in hemolymph in the stages ofPenaeus monodon ovary development. Experiment results show that, the concentrationof LH was the highest in vitellus accumulate phage (from Ⅱ to Ⅳ), in Ⅱ phage0.27mIU/ml and Ⅳ phage0.24mIU/ml. The concentration of LH was the lowest in ovarymature phage (Ⅴ)(0.10mIU/ml). The concentration of LH enlarged in recovery phage.The concentration peak of FSH (0.13IU/ml) was in initiation stage (Ⅱ) of ovarydevelopment and the density kept down in ovary and reached the minimum inⅤphage(0.03IU/ml). The concentration of FSH also enlarged in recovery phage. Theconcentration of P sustained development of all six stages and was the peak in Ⅵ phage(105.3pg/ml). The concentration of E2were virtually identical in all six stages and thepeak value (77.0pg/ml) in Ⅲ phage was1.2times of the least one (68.7pg/ml) inⅣphage, which showed a high level in six stages of ovary development, and displayedan alterative higher and lower trend. In conclusion, we conjectured that all of fourhormones had an important role in ovary development of the black tiger shrimp(Penaeus monodon) similar to mammalian.The technique of quantitative real time PCR (QRT-PCR) was used. For tissuesexpression profiles, there were9tissues which were ovary, lympho, neurosecretoryorgan in brain, blood, muscle, heart, gill, hepatopancreas and intestine. Thequantification result of three genes mRNA in9tissues of Penaeus monodon showed that,all the expression was different in different tissues. For ovary development expressionprofile, ovary tissues from Ⅱ phage to Ⅵ phage were detecded. The quantificationresult of cyclinB mRNA were in ovary the highest levels, followed by lympho and inhepatopancreas the lowest levels. The expression of cyclinB was significantly differentbetween ovary and other tissues by means of ANNON (P<0.01) and was nosignificantly different in ovary development stages. The quantification result of cdc25mRNA were in heart the highest levels, followed by muscle and in gill the lowest levels.The relative expression level of heart was886times compared with gill. And thequantification result of cdc25mRNA were in Ⅱ phage the highest levels, and in Ⅲphage and Ⅳ phage the lowest levels, with1.6times between Ⅱ phage and Ⅲ phage.The quantification result of PmMPP6mRNA were in lympho the highest levels,followed by muscle and in gill the lowest levels. The expression of PmMPP6wassignificantly different between all of tissues by means of ANNON (P<0.05). Thequantification results of PmMPP6mRNA were in Ⅱ phage the highest levels and in Ⅲ phage the lowest levels.