中华按蚊法尼酸甲基转移酶基因AsFAMeT的克隆、分子特征与表达模式分析OA

[Aim]This study aims to clone the farnesic acid O-methyltransferase gene(FAMeT)of Anopheles sinensis AsFAMeT,analyze its molecular characteristics,and investigate its expression patterns in different developmental stages and different adult tissues,thereby providing a theoretical basis for elucidating the biological functions and role of FAMeT in juvenile hormone synthesis pathway.[Methods]Based on the genomic data of An.sinensis,the full-length cDNA sequence of AsFAMeT of An.sinensis was cloned,and bioinformatic analysis was performed on the encoded protein.The pCold-TF-AsFAMeT recombinant plasmid was constructed and transformed into Escherichia coli BL21(DE3)receptor cells,and the recombinant protein was expressed by IPTG induction.The recombinant AsFAMeT was purified using a nickel column,and polyclonal antibody was prepared.The specificity of the antibody was detected by Western blotting.RT-qPCR was used to detect the expression levels of AsFAMeT in different developmental stages of An.sinensis(egg,1st-4th instar larvae,female and male pupae,and female and male adults)and tissues(head,thorax,abdomen,Malpighian tubules,fat body,midgut,ovary,testes and integument)of the 3-day-old female and male adults.[Results]The full-length cDNA sequence of AsFAMeT from An.sinensis was cloned(GenBank accession no.:PV368360),with an open reading frame of 1 050 bp in length,encoding 349 amino acid residues.AsFAMeT has the predicted molecular weight of 37.02 kD,an isoelectric point of 4.64 and no signal peptide sequence or transmembrane domain.AsFAMeT from An.sinensis shared high homology with FAMeTs from An.gambiae and An.darlingi,with the amino acid sequence identities of 77.71％and 69.43％,respectively.The prepared polyclonal antibody was able to specifically recognize the AsFAMeT protein.AsFAMeT was expressed in different developmental stages and various tissues of the 3-day-old female and male adults of An.sinensis.Higher expression levels of AsFAMeT were observed in the 4th instar larvae and female and male pupae of An.sinensis.The expression levels of AsFAMeT in the integument,ovaries and testes of the 3-day-old adults of An.sinensis were significantly higher than those in the other tissues,while those in the Malpighian tubules and midgut were relatively low.Western blotting protein quantification results were consistent with RT-qPCR results.[Conclusion]AsFAMeT of An.sinensis was successfully cloned and expressed in prokaryotes,and a specific polyclonal antibody was prepared,which can specifically recognize the natural AsFAMeT protein of An.sinensis.The expression patterns of AsFAMeT in different developmental stages and tissues of female and male adults of An.sinensis were investigated.The results provide an important basis for understanding the molecular mechanism of AsFAMeT in the regulation of growth,development and reproduction in An.sinensis.