| Bactrocera dorsalis(Hendel)is an important quarantine pest,which has complex feeding habits,wide host range,strong fertility,rapid propagation,strong adaptability,large potential distribution area and difficulty in control.Strong fertility is one of the important reasons for the continuous breeding of B.dorsalis.Therefore,the function of genes related to reproductive regulation was studied by means of molecular biology,which provided new targets and new ideas for the sustainable prevention and control of B.dorsalis.This study identified a proteasome Subunit beta type-3-like(pts)gene that was highly expressed in ovarian tissue during sexual maturation from the laboratory transcriptome library of B.dorsalis.Homologous comparisons and phylogenetic analyses were performed.The real-time fluorescence quantitative PCR and immunoblot techniques were used to define their temporal and spatial expression patterns.Then the functions of pts were used to research in ovarian development and fecundity of female adults using RNAi technology.It can provide a theoretical basis for revealing the molecular mechanism of pts gene regulating the reproductive development of B.dorsalis.1.pts gene identification and bioinformatics analysisWe identified pts gene that was highly expressed in the sexual maturation stage from the laboratory transcriptome library of B.dorsalis and obtained its full-length c DNA sequence with a size of 615 bp encoding 205 amino acids,and the coding regions were highly homologous with Bactrocera latifrons,Bactrocera oleae and Bactrocera cucurbitae,which were 97%,95%,and 92%,respectively,and were similar to Ceratitis capitata.2.The temporal and spatial expression patterns of ptsThe expression patterns showed that the expression level of pts gene were significantly higher in the egg and female sexual maturation stages than in larvae,pupal stages,and early eclosion stages of B.dorsalis.In ovary tissues of sexually mature adults,the expression level was significantly higher than that of other tissues by approximate four times.The expression of ovary on the 7th day after eclosion was significantly increased and maintained high expression.The expression levels of fat body of different days after emergence showed a tendency of gradual increase.In addition,the PTS protein content in ovarian tissues was significantly higher than that in other tissues,and the overall trend was consistent with the transcription level.3.Function analysis of pts gene in the reproductive development of B.dorsalis femalesIn order to further determine the functions of pts gene in the reproductive development of B.dorsalis,we observed the reproductive development-related behavioral characteristics of B.dorsalis after RNA interference.About 1/2 of female ovaries occurred to development retardation,which were significantly smaller than the normally developed ovaries.The average total egg production,daily egg production and female mating rate in the treated group decreased significantly,but there is no significant difference in egg hatching rate.To investigate the mechanism of pts gene,the expression levels of vitellogenin-related genes Vg1,Vg2 and yolkless were examined and it was found that all three genes were significantly down-regulated.The total proteins of ovary,fat body,and hemolymph were extracted on the 3rd day after interference and it was found that the expression of PTS protein was significantly decreased,at the same time the expression of Vg protein in the ovary was significantly decreased,while in fat body and hemolymph there was no significant change.These results indicate that silencing of the pts gene affects yolk deposition in the ovary without affecting the humoral transport process of the Vg protein.In summary,the pts gene may be involved in the regulation of ovary development and oviposition by affecting yolk deposition in female B.dorsalis,which in turn affects the reproductive development of females. |
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