Cloning Analysis Of MF Synthesis Pathway Gene And Protein Expression Analysis Of Juvenile Hormone Acid Methyl Farnesoate In Scylla Paramamosain

Scylla paramamosain(mud crab)is one of the main economic species in the southeastern coastal areas of China.Therefore,researchers are paying more and more attention to the crab,especially to the problems faced in the artificially cultured crab,include the small scale of cultivation,the low yield and early maturity of sexually mature female crabs,the high mortality rate and diseases from the zoea larva to the crab larvae.The study of crustacean endocrinology has important guiding significance for solving these problems.Methyl farnesoate(MF)is synthesized in the mandibular organ(MO),which is an important endocrine hormone regulating the growth,development and reproduction of crab,and plays an important role in the sexual maturation of crab.This thesis focuses on the vital hormone methyl farnesoate(MF),aiming at cloning of the genes involving in the synthesis of MF using RACE technology,discussing the roles of these genes during the development by virtue of qRT-PCR method.The protein expression anaysis of juvenile hormone acid methyl transferase(JHAMT)was also studied to provide some important theoretical basis for solving the problems in artificial culture of Scylla crab.The main research results are as follows:1.Cloning and Expression Analysis of FPPS,FoD and FaD Genes in S.paramamosain.Fanesyl diphosphate synthases(FPPS),farnesyl dehydrogenase(FoD)or farnesyl dehydrogenase(FaD)are key regulators of MF synthesis in crustaceans.Fanesyl diphosphate(FPP)was synthesized by multi-step reaction catalyzed by FPPS,and farnesyl diphosphate(FA)was synthesized from farnesyl alcohol by FoD and farnesyl aldehyde by FaD.The sequences of these three genes were obtained and named Sp-FPPS,Sp-FoD and Sp-FaD,respectively.The full length of Sp-FPPS cDNA was 1548 bp,including 1290 bp ORF encoding 429 amino acids.Sequence analysis reveales that there are two highly conserved aspartic acid motifs in the domain containing pyrophosphate synthase,which might be related to the functional sites of the enzyme.The expression analysis showed that the changes of Sp-FPPS were very significant in zoea 3th(Z3),Z5 and megalopa.In adult tissues,the highest expression was found in MO,followed by muscle,suggesting that the function of Sp-FPPS is closely related to individual development,especially larval development.The expression of Sp-FPPS was significant in the fourth,fifth and sixth stages of ovarian development,and the content of MF synthesized by crabs was relatively high.Therefore,it was speculated that Sp-FPPS was related to the synthesis of MF.Sp-FoD is 1548 bp in length with an ORF of 1137 bp encoding 378 amino acids.The 5’UTR and 3’UTR are 152 bp and 980 bp,respectively.The predicted molecular weight of the protein is 40 kD,with an isoelectric point of 6.71.It was predicted that there was no signal peptide,not a secretory protein.Alcohol dehydrogenase activity site and NAD binding domain were found in the sequence.The gene sequence is highly conserved and similar to function of other species.The expression of Sp-FoD showed that the changes of Sp-FoD were very significant in the Z3,Z5 and M of larvae development,and the highest expression in tissues was in muscles,followed by in epidermis.The expression pattern of Sp-FPPS was similar to that of Sp-FPPS in different ovarian development stages.It is inferred that the time expression of Sp-FPPS and Sp-FPPS is identical.The full length of Sp-FaD is 1749 bp,and the ORF was 1506 bp.It encoded 501 amino acids.The 5’UTR and 3’UTR were 142 bp and 101 bp,respectively.The predicted molecular weight of the protein is 56 kD and the isoelectric point is 6.2.It was predicted that there was no signal peptide.The qPCR results analysis showed that the changes of Sp-FaD were very significant in Z2,Z3 and Z5 of the larvae,and the expression of Sp-FaD in adult tissues was the highest in maxillary organs,especially in stages 4 and 6 of ovarian development.2.Protein analysis of juvenile hormone acid methyltransferase in S.paramamosain.Juvenile hormone acid methyltransferase(JHAMT)is a key enzyme in JH synthesis pathway and a member of the methyltransferase family in arthropods.It speculated that it may be a key enzyme for MF synthesis in crustaceans.The recombinant expression vector of pET28a-SpJHAMT was constructed and transformed into the expression strain.The recombinant protein SpJHAMT was identified,purified,renatured and functional validated.In conclusion,three enzymatic genes in the MF synthesis pathway were preliminarily studied,which indicated that their expression might be related to the synthesis of MF.The vector of JHAMT,the last step in the synthesis of MF,was successfully constructed and the recombinant protein was obtained to prepare for its functional verification.It laid a foundation for further study on the synthesis mechanism of MF.