Full-Length Cdna Cloning And Spatio-Temporal Expression Analysis Of Juvenile Hormone Epoxide Hydrolase (JHEH)Gene In Macrobrachium Nipponense

Oriental river prawn, Macrobrachium nipponense, is considered as a major freshwater economic aquaculture species of shrimps. Oriental river prawn culture, an increased economic value species for farmers, plays an important role in aquatic products culturing in our country. Rapid development of oriental river prawn culture in big scales carried out many problems, such as reverse selection and reproduction by close genetic descent due to the usage of the remaining prawns as parents for the next year, resulting in degeneration of the species. The key characteristics are as follows:sexual prematurity, short molting period and early molting of procreation etc, resulting in the small size and low economic value of the mature prawns, thus severely restricted the development of oriental river prawn culture. Like other crustaceans M. nipponense has periodic molting characteristic. Juvenile hormone epoxide hydrolase (JHEH) is an important enzyme responsible for decomposing juvenile hormone (JH), playing a key role in growing, molting and reproducing processes according to the researches of that in insects. However researches on JHEH gene have not been reported in crustacean species.In this research, the full-length cDNA sequence of JHEH gene was firstly cloned from the testis of oriental river prawn using rapid-amplification of cDNA ends (RACE) technique, based on the middle sequence from the high-quality testis cDNA library of oriental river prawn. The full length cDNA sequence of JHEH gene was1848bp, containing a184bp5’ untranslated region (UTR), a281bp3’ UTR and a1383bp open reading frame (ORF), encoding460amino acids with molecular weight of51.143kDa and theoretical isoelectric point of6.43. The upstream region of poly (A) has a significant polyadenylation signal AATAAA. Amino acid polypeptide precursor includes signal peptides containing22amino acids and mature peptides containing438amino acids. There is a conserved catalytic center domain of Asp-His-Asp which reveals a high level of similarity to the epoxide hydrolase superfamily and belongs to the a/p hydrolase.The comparison of JHEH peptides with the Tribolium castaneum, Bombus impatiens, Harpegnathos saltator, Glossina morsitans, Culex quinquefasciatus and Bombyx mori revealed that the amino acid similarity is over95%and the identity is over40%, suggesting JHEH gene is highly conversed in the evolution of Arthropoda. According to the analysis of the Phylogenetic Tree, crustaceans and insects are separatly assigned into different branches, predicting crustacean and insect has long genetic distance. Interestingly, M. nipponense assigned into the same branch with dipster, coleopteran and Lepidoptera and then assigned into the same branch with hymenopteran.The expression level of JHEH gene in various tissues of M. nipponense and time-dependent pattern after embryo was detected by Quantitative real-time PCR (qRT-PCR) for the first time. According to the analysis results, JHEH gene has the highest expression level in hepatopancreas, while lowest in muscle. The expression levels in turns as follow:hepatopancreas> intestine> vental nerve cord> ovary> testis>eyestalk> brain> hear> muscle, which is similar to the expression pattern in many other species. The result of time-dependent pattern showed that JHEH gene had the highest expression level in1-day age shrimp which is very significantly different from other periods (P<0.01). The JHEH gene had the second highest expression level in10-day age, and there is no significant difference between20-day age and30-day age. At last, the research on expression levels of JHEH gene in M. nipponense suggested that the expression level in epidermal muscle in molting period is higher than that in non-molting period and the difference is significant, however, the expression level in brain in molting period is lower than that in non-molting period.It is the first time to clone the full length cDNA sequence of JHEH gene of oriental river prawn and analysed the structure of JHEH gene in our research. Besides, qRT-PCR technique was firstly used to detect the expression level of JHEH gene in various tissues and periods after embyro. This research provides basic information for the function of JHEH gene, and promotes further studies in the process of molting and reproduction of oriental river prawn, as well as providing references for resolving bottleneck problems which restrict the development of oriental river prawn culture, such as sexual prematurity.