Cloning And Functional Analysis Of Carboxylesterase Gene Bmbe2in The Silkworm, Bombyx Mori

Carboxylesterases (COEs) are a class of poly-proteins, which catalyze thehydrolysis of esters, sulfate esters and amides, and are widespread in all livingorganisms, including plants, animals, insects, microbes, and also belong to be amultifunctional superfamily. COEs play different roles in xenobiotic detoxification,neurogenesis and pheromone degradation. In addition, COEs have reproductive anddevelopmental regulation roles in insect. Previous studies have shown that Esterase-6expressed in male accessory glands of Drosophila melanogaster is a member ofcarboxylesterase family, which can be transferred to the female reproductive tractduring mating, thereby affecting the eggs-laying and mating behavior of femaleDrosophila. However, it is unclear whether the enzyme has similar function in otherspecies. Silkworm carboxylesterase family has been identified. The results indicatedthat Bmbe2was specifically expressed in MAGs. Thus, Bmbe2may have similarfunction to Drosophila EST-6, but it remains to be confirmed.In this study, I cloned Bmbe2, investigated expression pattern of Bmbe2and itsbehavior effects by RNAi. The results are as follows.1) Molecular cloning and sequence analysis of Bmbe2geneThe CDS length of Bmbe2is1843bp. Bmbe2encodes570amino acids, itsmolecular mass and isoelectric point are66.4kD and5.42, respectively. It potentiallyhas1N-glycosylation site and41phosphorylation sites. Signal P4.0analysis revealedthat Bmbe2probably has a signal peptide with32amino acids. Therefore, this proteinmay be a secreted protein. The results of phylogenetic analysis and sequence similarityanalysis showed that Bmbe2belongs to be β-esterase family, and Bmbe2is the putativeortholog of Hacarboxyl/cholineesterase with71.9%amino acid identity.2) Expression pattern of Bmbe2geneThe expression pattern indicated that Bmbe2gene has a high expression insilkworm larvae and adult antennas, foot, head of the jaw and is also highly expressed inthe adult male accessory gland, suggesting that Bmbe2may be related to sex behavior.3) Functional anaylsis of Bmbe2geneAccording to Bmbe2CDS, we designed three pieces of interference primers with700bp length to amplify, covering the entire coding sequence. With PromegaRiboMAX TM Large scale system a T7kit, we obtained dsRNA comprising a T7 promoter, and then injectied male silkworm pupae abdomen with a micro syringe.Real-time quantitative RT-PCR analysis suggested that RNAi significantly reduced theamount of Bmbe2gene expression in male accessory glands of silkworm moths. RNAimale silkworm moths crossed with normal female silkworm moths significantly reducedthe rate of laying eggs of female moths, and the females prefer to accept the mating inre-mating experiments.In summary, we cloned the Bmbe2gene and investigated the expression pattern ofBmbe2gene and the mating behavior effect of Bmbe2by RNAi. I confirmed thatBmbe2can increase the rate of eggs-laying of female moths by mating transfer anddecrease receptivity to remating of female moths. The results presented in this studyprovide useful function information for Bmbe2.