| Black tiger shrimp (Penaeus monodon) is regards as one of the largest businessculture shrimps, and also is an important tranditional cultivation breed in China.Because of the mechanism of propogation and breed of Penaeus monodon is not clear,eyestalk ablation is still commonly practiced in shrimp to induce ovarian maturation incaptivity. While the ablation can induce ovarian maturation, it also jeopardizes growth,shortens molting cycle, increases energetic demands, and resulting in an eventual loss inegg quality and high mortality. So it is very necessary to study the mechanism ofgonadal development and develop new effective ripened technology, which caneffectively improve the utilization and survival rate of broodstock and save cost, withthe result of the steady development of shrimp industry. In this study, two functionalgene correlated with the regulation of gonadal development—adenine nucleotidetranslocase and nm23was cloned, and its expression mechanism is preliminarilyexplored. The study offers some basic referrence for further research about geneexpression concerned with gonadal development.1. In the present study, the full sequence of P. monodon ANT gene was cloned andnamed PmANT. The full length cDNA of PmANT contained a5’ untranslated region(UTR) of65bp, a3’ UTR of393bp and an ORF of930bp encoding a polypeptide of309amino acids with an estimated molecular mass of33.622kDa. As other animalANTs, the structure of the PmANT protein consists of three homologous repeateddomains. But there are not signal peptide and glycosylation sites in PmANT protein.Sequence alignment analysis showed that the PmANT and the ANT of Litopenaeusvannamei shared a similarity of98.7%and the homology of97.4%. Analysis of thetissue expression pattern of the PmANT showed that the PmANT mRNA was expressedin all tested tissues, including heart, ovary, testis, muscle, eye disease nerve, cranialnerve, lymphoid tissue, stomach, intestines, hepatopancreas, gill, hemocyte, with thehighest levels in muscle of male and the lowest level in immature testis. Furthermore,the PmANT expression was found to be a higher level in mature testis than in immature testis. The PmANT expression was found in the six ovarian stages of development andthe expression of ovarian growth levels in the third growth phase was significantlyhigher than that of others except the sixth growth phase, while lowest in the fourthgrowth phase. The study provided essential materials and laid a firm foundation for thefurther research of the function of PmANT in gonad development of P. monodon.2. The techniques of Rapid Amplification of cDNA Ends were used to clone theP.monodon nm23gene. The complete gene sequence of nm23was consisting of769nucleotides, the5’-UTR length among which is105bp,3’-UTR length is208bp, theORF region is456bp, encoding corresponding151amino acids with an estimatedmolecular mass of17.01kDa. The nm23protein of P.monodon possesses uniquewell-conserved NDP kinase active site motifs as other nm23family members. And wefoud that there are not signal peptide and glycosylation sites in nm23protein, usingSingalP4.0program and the online program NetNGlyc1.0. Sequence alignmentanalysis showed that the nm23and the nm23of Litopenaeus vannamei shared asimilarity of98.7%and the homology of97.4%. Analysis of the tissue expressionpattern of the nm23showed that the nm23mRNA was expressed in all tested tissues,including heart, ovary, testis, eye disease nerve, cranial nerve, lymphoid tissue, stomach,intestines, hepatopancreas, hemocyte, with the highest levels in eye disease nerve offemale, followed by immature testis, and there was significantly difference with theother organizations. Furthermore, the nm23expression was found to be a higher level inimmature testis than in mature testis. The nm23expression was also found in the sixovarian stages of development and the expression of ovarian growth levels rosegradually from the one growth phase to the fourth growth phase, and then reduced bydegrees. So we speculate that the P.monodon nm23gene may play a driving role inovarian development. The cDNA fragment encoding mature protein of nm23wassubcloned to expression vector pRSET and transformed into Escherichia coli BL21.The recombinant nm23(Rnm23) was expressed and purified under optimizedconditions and it provided a basic material for studying the function of nm23in gonaddevelopment. |
