中华豆芫菁短链脱氢酶基因EcDHRS4的分子特征及功能分析OA

[Aim]To analyze the molecular characteristics of the dehydrogenase/reductase SDR family member 4(DHRS4)gene in Epicauta chinensis(EcDHRS4),clarify its expression pattern,and explore its influence on cantharidin synthesis in E.chinensis.[Methods]The full-length cDNA sequence of EcDHRS4 in E.chinensis was cloned by RT-PCR and RACE technologies,and its sequence characteristics were analyzed.The three-dimensional structure of EcDHRS4 was modeled by homology modeling.The expression levels of EcDHRS4 in E.chinensis adults on day-1,3 and 5 after mating were detected by RT-qPCR.dsEcDHRS4 was injected into male adults of E.chinensis immediately after mating for RNAi,and the relative content of cantharidin was measured by gas chromatography on day-1,3,5 and 7 after injection.[Results]The open reading frame of EcDHRS4 of E.chinensis(GenBank accession no.:MW282959)was 807 bp in length,encoding 268 amino acids with the predicted molecular weight of 28.71 kD and isoelectric point(pI)of 9.30.EcDHRS4 was a hydrophilic and stable protein,lacking a signal peptide and transmembrane domains.The secondary structure of EcDHRS4 was mainly composed of α-helices and random coils.EcDHRS4 belonged to the classical SDR family,and the sequence similarity among different SDR members within the same species was low.There was significant difference in the expression level of EcDHRS4 between different genders of E.chinensis,and the expression level of EcDHRS4 in male adults reached the peak on day-3 after mating.The expression levels of EcDHRS4 were down-regulated by 98.30％,99.09％,84.15％and 91.79％on day-1,3,5 and 7,respectively,after dsEcDHRS4 injection,as compared with those of the dsGFP control group,and the relative content of cantharidin decreased to 56.81％of that of the dsGFP control group on day-7 after injection.[Conclusion]EcDHRS4 belongs to the C1-SDR family protein.The expression levels of EcDHRS4 in E.chinensis adults at different time after mating show significant gender differences,and EcDHRS4 is highly expressed in male adults after mating.Inhibition of EcDHRS4 expression affects the synthesis of cantharidin,indicating that EcDHRS4 is involved in the biosynthesis of cantharidin.These findings enhance the functional understanding of insect SDR genes,and lay a foundation for further elucidating the role of EcDHRS4 in the biosynthesis of cantharidin.