Identification And Expression Analysis Of The FAMeT Gene,the Rate Limiting Enzyme Of Methyl Farnesoate Biosynthesis Pathway In Capitulum Mitella

The Capitulum mitella is a natural marine treasure and a special marine economic species in China.Due to the scarcity of resources caused by over-exploitation,it is urgent to carry out research on large-scale artificial hatchery of C.mitella and build a hatchery breeding and control system.The group has already confirmed the important function of juvenile hormone in the metamorphosis reaction of C.mitella cyprid larvae during the actual seedling breeding process.Therefore,it is important to investigate the synthesis and regulation of the crustacean hormone methyl fanesoate(MF)for the hatchling breeding of C.mitella.In this paper,we used bioinformatics methods to identify the farmesoi acid O methyl transferase gene(FAMeT),the rate-limiting enzyme of MF synthesis pathway in C.mitella,and carry out bioinformatics analysis,based on the genomic data of C.mitella assembled by the group in the early stage.By studying the difference of expression levels at different stages,analyze the function of the CmFAMeT1 gene in the growth and metamorphosis of C.mitella.We also cloned the full sequence of its coding region,constructed a recombinant plasmid to express CmFAMeT1 recombinant protein,and analyzed the recombinant protein activity in vitro,in order to reveal the function of CmFAMeT1 in the MF synthesis pathway and to provide a reference for discussing the growth and metamorphsis mechanism of C.mitella.Research results:(1)Using DEGseq2 software to screen a large number of differential genes at C.mitella different developmental stages,we indicated the C.mitella has different gene expression profiles in each developmental stage.Six CmFAMeTs gene were identified from the C.mitella genome using NCBI,Pfam database,bidirectional BLAST,and predicting the subcellular localization,transmembrane structure,signal peptide,primary,secondary and tertiary structures of amino acid sequences of these six gene,also the chromosomal localization of these gene.The physicochemical properties varied greatly among the six members,with isoelectric points 4.91-8.30;encoded amino acid number of 290-1758;molecular weight 32.44 k Da-199.45 k Da;CmFAMeT1 and CmFAMeT2 are stable protein,and CmFAMeT3,CmFAMeT4,CmFAMeT5 and CmFAMeT6 are unstable protein;all six protein are hydrophilic protein.The amino acid motif composition encoded by members of the CmFAMeT gene family lacks motif2 and motif5 compared to other crustaceans;CmFAMeT1,CmFAMeT2,CmFAMeT4 and CmFAMeT6 are localized outside the cell,CmFAMeT5 is localized at the plasma membrane,CmFAMeT3 may be located outside the cell or at the cytoplasmic matrix;CmFAMeT1,CmFAMeT2,and CmFAMeT3 have no signal peptide structure,while CmFAMeT4,CmFAMeT5,and CmFAMeT6 have signal peptides;CmFAMeT5 is a transmembrane protein;the number of phosphorylation and glycosylation sites also varies widely,and the randomly coiled and extended chains are the main components of the secondary structure of CmFAMeT gene family members.CmFAMeT5 is located on chromosome 13;CmFAMeT1 and CmFAMeT3 are located on chromosome 8,and CmFAMeT2,CmFAMeT4 and CmFAMeT6 form a distinct gene cluster located on chromosome 15.Phylogenetic analysis of the full amino acid sequences showed that the six CmFAMeTs gene family members were distributed in the insect cluster,while CmFAMeT1 clustered into a separate group in the insect clusters,presumably with functions that may be both similar to and distinct from those of insect FAMeT.The homology of Methyltransf＿FA sequence of CmFAMeT1 was compared with that of crustaceans,and it was found that CmFAMeT1 had higher homology with Methyltransf＿FA sequence of other crustaceans,and it was speculated that its function might be conserved in crustaceans.(2)The screening of internal reference genes was carried out by conventional PCR electrophoretic bands observation,lysis curve specificity and expression profile analysis,and Norm Finder stability assessment.Selecting 60 s ribosomal protein L15(rp L15-0390)as the internal reference gene for studying the expression of CmFAMeT1 gene in different developmental stages of the C.mitella.The CmFAMeT1 gene was highly expressed in the mature stage ovaries of the C.mitella,suggesting that the CmFAMeT1 gene plays an important role in C.mitella ovary development;The expression level of CmFAMeT1 gene showed no significant(P>0.05)changes in embryonic stage,stage II of nauplius and stage IV nauplius;during the process from stage VI nauplius to the metamorphosis of intra-membranous stage into cyprid,CmFAMeT1 expression showed a significant(P<0.05)up-regulation trend,indicating that the increase of CmFAMeT1 expression level promoted the synthesis of MF and induced the metamorphosis of C.mitella into cyprid.After metamorphosis into cyprid,CmFAMeT1 expression level was significantly(P<0.05)down-regulated continuously,and CmFAMeT1 expression at cyprid 5 days was not significantly(P>0.05)different from that of stage II and stage IV nauplius larvae without metamorphosis,and CmFAMeT1 expression levels were significantly(P<0.05)up-regulated at the early,mid and juvenile stages of metamorphosis after artificial induction,which was higher than that of cyprid for 5 days.CmFAMeT1 promotes the synthesis of MF by farmesoic acid(FA),and the increased synthesis of MF may promote the developmental metamorphosis of cyprid larvae into juveniles.(3)According to the genomic sequence information,the full-length CDS sequence of CmFAMeT1 gene was cloned and connected to PMD19-T vector,transferred into DH5α strain,and sequenced by picking a single clone to verify that the sequence information was consistent with the genomic information,and the coding region of the CmFAMeT1 gene was 873 bp in length,encoding 290 amino acids.After the CmFAMeT1 gene was obtained,the p ET28a-CmFAMeT1 recombinant plasmid was constructed by inserting it between the Bam HI and Xho I digestion sites of the p ET28 a vector,and the CmFAMeT1 protein expression was induced by adding IPTG.The CmFAMeT1 recombinant protein was efficiently expressed in the p ET28a-CmFAMeT1 recombinant expression vector,but most of the CmFAMeT1 recombinant protein appeared as inclusion bodies.(4)The induction temperature,concentration and inducer species of CmFAMeT1 recombinant protein expression were optimized to reduce the proportion of inclusion bodies,and the optimal temperature suitable for CmFAMeT1 recombinant protein expression was determined to be 24°C,the optimal inducer was IPTG,and the optimal induced IPTG concentration was 0.5 m M.The induction under these conditions for 10 h successfully increased soluble expression of the CmFAMeT1 recombinant.Using the nickel column,the recombinant protein was screened by different concentrations of imidazole,and it was found that the 30 m M imidazole eluate had the strongest elution ability.After the eluate was dialyzed,the recombinant protein was concentrated by a 10 k Da size concentration tube,and the concentration of recombinant protein was 28 mg/ml measured by Broadford method,and a higher yield of CmFAMeT1 recombinant protein was obtained.The in vitro enzymatic activity assay system of CmFAMeT1 recombinant protein was constructed,i.e.50 μg FA was added to 450 μl of Tris.HCl(50 m M,Ph 7.5)buffer containing 1.1 m M SAM,and 28 μg CmFAMeT1 recombinant protein was added after incubation at 25℃ for 5 min,incubated for 30 min,and the reaction products were detected by GC-MS after termination of the reaction.The substrate FA was detected in both the experimental group and the control group,and the peak area of FA in the experimental group was 26.17% less than that of the control group,but MF was not detected.In this study,members of the C.mitella FAMeT gene family were analyzed using bioinformatics methods in order to lay the foundation for an in-depth study of the biological functions of C.mitella FAMeT.The expression levels of CmFAMeT1 gene in different developmental stages of the C.mitella were investigated,revealing that FAMeT plays an important role in the development of the C.mitella ovary,and in the two metamorphic processes from nauplius larvae to cyprid larvae and cyprid larvae to juveniles,indicating that MF is closely related to the regulation of growth and metamorphosis in C.mitella larvae.After cloning CmFAMeT1,a recombinant plasmid was constructed and induced expression in a prokaryotic expression system to obtain soluble CmFAMeT1 recombinant protein,which provided material for the functional study of CmFAMeT1 and laid the foundation for further revealing the MF synthesis process.