| Bactrocera dorsalis(Hendel),is the world-wide quarantine pest,has a wide range of hosts and strong reproductive capacity,and it is difficult to control.After emergence,the adult must undergo a period of time to reach sexual maturity and then mate to lay eggs,so the sexual maturity process is also an important entry point for pest control.Therefore,studying the molecular mechanism of the reproductive development process of female adult can not only enrich and improve the insect reproduction regulation mechanism,but also provide new ideas and directions for the green and sustainable management of B.dorsalis.We screened 12 miRNAs highly expressed in female adults from our previously established miRNA library,including 8 known miRNAs and 4 novel miRNAs.Real-time PCR(qRT-PCR)was used to detect the expression pattern of different reproductive development stages in ovary and fat body of female adults;the novel miRNA pc-3p-116 was selected due to its specific expression in ovary and differential expression during different reproductive developmental stages.The function of miRNA pc-3p-16 in ovary development and fecundity was detected by micro-injection.The molecular mechanism of regulating the reproductive development of female adults was determined by target gene prediction,dual luciferase assay and RNAi.1.Identification of differentially expressed miRNAs and temporal-spatial expression pattern in reproductive development of B.?dorsalisWe screened 12 miRNAs highly expressed in female adults from our previously established miRNA library,including 8 known miRNAs and 4 novel miRNAs.Real-time PCR(qRT-PCR)was used to detect the expression pattern of different reproductive development stages in ovary and fat body of female adults.The results showed that 6 miRNAs were differentially expressed in different reproductive development stages of female adults.Among them,the expression of pc-3p-116 in ovary and fat body showed a downward trend during maturity.let-7-5p,miR-34-5p,miR-263a-5p,pc-3p-1934 and miR-2b-3p were highly expressed in the sexually mature female adults(4d-13 d post emergence),indicating that these six miRNAs are involved in the regulation of the reproductive development of female adults.We further examined the expression patterns of seven miRNAs in different tissues of the female adults 1d and 13 d post emergence,respectively.The results showed that pc-3p-116 had highly abundan in the ovary,and the expression level was significantly decreased with ovary development.let-7-5p was expressed lowly in the head and ovary,and the other four miRNAs were expressed in different tissues.Our results suggest that pc-3p-116 is involved in the regulation of reproductive development of female adults.2.Functional study of miRNA pc-3p-116The pc-3p-116 agomir was injected into the female adults by micro-injection techniques to detect its function in reproductive development.The results showed that there was significant developmental delay in the ovary of the treatment group.The total number of eggs laid during the 15 days after mating(353 eggs)was significantly lower than that of the control group(700 eggs),and the average daily egg production was lower than that of the control group.There was a significant difference in the number of single female adult eggs at 3d,5d,7d,9d,11 d and 13d;the hatching rates of the treatment group and the control group were 55% and 75%,respectively,with significant differences.The above results indicate that pc-3p-116 is involved in the regulation of ovary development and fertility in B.dorsalis.We used TargetScan and miRanda software to predict pc-3p-116 target genes,and selected two software-predicted intersections to obtain 6 potential target genes,including cytochrome P450 family gene CYP6a14,Cullin family gene cullin2,and E3 protein ligase E3 SUMO-protein ligase PIAS2,nuclear hormone receptor gene FTZ-F1,Endophilin family gene Endophilin B1,nuclear receptor coactivator coactivator2.Real-time quantitative PCR was used to detect target gene expression after injection of pc-3p-116 agomir,the results showed that CYP6a14,Endophilin B1,PIAS2 and cullin2 showed a significant decrease in the treated group compared with control group after injection of the pc-3p-116 agomir.The expression levels of CYP6a14 and Endophilin B1 were significantly up-regulated in the sexual maturity stage of female adult,which was contrary to the expression pattern of pc-3p-116.Next,the 3’UTR region of potential target genes CYP6a14 and Endophilin B1 were inserted into the psiCHECK-2 vector to construct a dual luciferase reporter vector,and were co-transfected with the miRNA agomir into the HEK293 T cells line.In the double luciferase assay,the results showed that the dual luciferase reporter vector constructed by the two genes CYP6a14 and Endophilin B1 significantly reduced the luciferase signal when co-transfected with pc-3p-116 agomir,indicating that these two genes are potential target genes of miRNA pc-3p-116.In order to further study the molecular mechanism of miRNA pc-3p-116 in the reproductive development of female adults,according to CYP6a14 and Endophilin B1 3’UTR binding site specificity and binding free energy with pc-3p-116,we chose to study the function of its potential target gene CYP6a14 in female adults.In order to further clarify the function of CYP6a14,we used RNAi technology to silence CYP6a14 of B.?dorsalis,and detected a series of reproductive behaviors such as ovary morphology,mating rates,fecundity and hatchability,after interfering with CYP6a14 by micro-injection.The results showed that there was significant developmental delay in the ovary of the treatment group.The total number of eggs laid during the 15 days after mating(218 eggs)was significantly lower than that of the control group(340 eggs),and the average daily egg production was lower than that of the control group.There was a significant difference in the number of single female adult eggs at 3,5,7,9,11,13 and 15 days;the hatching rates of the treatment group and the control group were 60% and 80%,with significant differences.The above results indicate that CYP6a14 is involved in the regulation of ovary development and fertility in B.dorsalis.In summary,pc-3p-116 affects the reproductive behavior of Bactrocera dorsalis by regulating the gene CYP6a14. |
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