Cloning And Functional Analysis Of Sex Determining Genes,dsx And Tra2 In Bactrocera Cucurbitae

Bactrocera cucurbitae Coquillett is a quarantine pest of entry plants in China,against more than 120 kinds of vegetables and fruits such as Cucurbitaceae,Solanaceae,papaya,and guava,caussing huge economic losses in Guangdong,Guangxi,Hainan and other places.Pesticide control is the main way to prevention at present,but biological control is the development trend of research.Therefore,this paper mainly studies the sex determination pathway of B.cucurbitae about the dsx gene and tra2 gene to make a theoretical basis for producting a large number of males.In this paper,sequences of dsx gene and tra2 gene of B.cucurbitae were determined;and the expression levels of the two genes in different developmental stages were clarified;the expression levels of the two genes after feeding dsRNA were initially explored.In this paper,the cDNA fragments of the dsx gene and tra2 gene of B.cucurbitae were obtained by cloning and both contained ORF.The dsx was transcribed sex-specifically:the open reading frame of dsx-f was 969bp in length,encodes a polypeptide of 322 amino acids,while the open reading frame of dsx-m was 1206bp in length,encodes a polypeptide of 401 amino acids.Both the dsx-f and dsx-m are including two high conservation regions,DM domain and dsx-Dimer.No sex-specific transtript was detected in tra2.The open reading frame of tra2 was 750bp in length,which encodes a polypeptide of 250 amino acids.It belongs to SR proteins family,with an RRM domain and RS regions at both ends.Quantitative detection of dsx gene and tra2 gene expression at different development stages of B.cucurbitae by qPCR technique showed that,the same dsx gene fragment of male and female adults of B.cucurbitae expressed the highest expression at the age of the instar larvae.The 1d male adults was significantly higher than that of the 1d female adults.The dsx-f was the lowest in eggs at 2-4 h,and the expression level was the highest in 1d females.The dsx-m was the lowest in eggs at 2-4 h,and the highest in instar larvae.The tra2 gene was expressed higher in the embryo stage and had the characteristics of maternal genetic expression.The expression level of female adults at 1d was significantly higher than that of adult male at 1d.Feeding dsRNA could affect the expression of dsx and tra2 genes in B.cucurbitae,but the silencing effect was not consistent with feeding 200μL 1μg/μL dose of dsRNA to B.cucurbitae adults.When dsx-dsRNA was fed,dsx expression in adult females increased while male adult expression decreased.Feeding tra2-dsRNA increased the expression of tra2 in female and male adults.