| The oriental fruit fly, Bactrocera dorsalis (Hendel), is a destructive pest for lots of fruits and vegetables. It’s distribution is widely and gradually became one of the most important pests to citrus. This insect has vigorous reproductive capacity and lay eggs into fruit and the developing larvar tunnel through the fleshy mesocarp on which they feed, and thus leading the abscission of fruits. Typically, the chemical pesticide is the main method to control B. dorsalis. According to the increasing reports of the resistance to pesticides, it is emergency to investigate new and safe pest management strategies in order to continuely control the oriental fruit fly. Ovary is an important reproductive system in insect, which is directly related to reproduction. The regulation to ovary development is closely involved in insect population development. Research onthe related aspects is becoming increasingly popular. Meanwhile, the ovary regulation is an important method in pest control. Based on the B. dorsalis ovary transcriptomic, the molecular characteration and function identification of the genes related to ovary development were investigated in this research, inthe hope of exploring new pesticide based on the regulation of ovary development. Main contents are as follows:1. Transcriptional analysis to B. dorsalis ovary and qPCR detection of the genes involved in ovary developmentThe transcriptional information from organisms can be obtained rapidly and accurately via transcriptome. The cDNA library was constructed after the total RNA was exacted from different days of ovary and sequenced in Illumina Hiseq platform. As a result, a total of 44,845 contigs and 23,822 unigenes were obtained. The mean length of unigene is 1,116 bp, which presented that this transcriptome has high quality. Based on the cut-off E-value of 10-5, there were 15,473 unigenes successfully annotated into Nr database,12,603 into Swiss-Prot,11,068 into KEGG,6,102 into COG and 11,043 into GO database, respectively. The homology analysis of the unigenes annotated suggested that the unigenes were mainly concentrated in Drosophila (79.5%), in which the highest homology relation was Drosophila virilis (13.68%) and the second one was Drosophila melanogaster (11.87%).The result of GO classification showed that 11,043 unigenes were categorized into GO database. Among the 59 functional categories, the "cellular process" "single-organism process" and "cell part" were the most three ones, contained 8,227 (74.5%),7,228 (65.5%) and 6,655 (60.3%) unigenes. The categoties, in which the unigenes were in the minimum number, were "protein tag" and "receptor regulator activity", all of which contained 1 unigene. These two categories were belonged to "Molecular function". After the COG alignment to unigenes, the results showed that 6,102 unigenes were successfully annotated in COG database, "general function prediction" contained 2,653 unigenes, which is the most abundant one, while the "nuclear structure" contained only 2 unigenes. The results of Go and COG preliminarily showed the distribution of gene function in B. dorsalis ovary transcriptomeAfter the analysis of transcriptome,11 pathways and 30 genes were isolated from the database based on their relation with ovary development. Among them,2,2,2,4,5, 2,5,4,2 and 1 genes belongs to Bone morphogenetic protein signaling pathway, E-cadherin pathway, Notch pathway, piRNA pathway, insulin signaling pathway, JAK-STAT pathway, Ecdysone signaling pathway, fat digestion and absorption pathway, vitellogenesis formation process, polar cell formation process and trehalose metabolism process, respectively. Afterwards, expression patterns of 30 genes in different segments and tissues were detected by qPCR. The results revealed that BMP pathway genes were mainly expressed in midgut; E-cadherin and JAK-STAT pathway genes were mainly expressed in Malpighian tubule; Notch, piRNA pathway and the polar formation genes were mainly expressed in ovary; insulin and ecdysone signaling pathway genes were equally expressed in all the tissues, while fat digestion and absorption pathway and vitellogenesis genes were mainly expressed in fat body. The results of qPCR preliminarily indicated the possible function of these pathway genes, which were meaningful to the identification of gene function.2. The cloning and sequence analysis of the genes related to piRNA pathway of B. dorsalisBased on the sequence information of the genes related to piRNA pathway in ovary transcriptome, the full length of genes were obtained by PCR. The open reading frame of genes piwi、argonaute3、 aubergine and tudor were 2,631,2,703,2,667 and 8,109 bp, which encodes 876、900、888 and 2,702 amino acids, respectively. After analyzingthe functional domains of these sequences, the results showed that all the deduced amino sequences were not transmembrane proteins and Bdpiwi, Bdago3 and Bdaub both contain a PAZ domain and a PIWI domain. Among these domains, there are 7 and 4 active sites, respectively. The PIWI domain contains 5’RNA guide strand anchoring site. These results revealed that these genes are possibly clossely related with cleavege of RNA in the piRNA formation. The Bdtud contains 10 TUDOR domains and 2-10 domains contain 4 methylation of arginine binding site, which indicated that Bdtud has the ability to bind with PIWI family proteins.The results of phylogenetic analysis of Piwi, Ago3 and Aub in insect showed that Piwi and Aub were categorized into one cluster, indicating a higher homology between these two genes. In addition, Ago3 was categoried into another cluster, indicating a deeper degree of differentiation of this gene. The Tudor sequence and 3 related homolog proteins:Spindle-E, Tudor-SN and Partner of PIWI were selected from NCBI for the phylogenetic analysis. The results showed that the Partner of PIWI were near to Tudor in relationship, while Spindle-E were farthest to Tudor. These analyses indicated the classification of the protein function.3. RNAi to the gene related to piRNA pathwayWith utilizing RNAi technologies, the function of Bdtud was investigated. The silence efficiency were not significant in whole body but in ovary, while a significant lower expression occurred in Bdpiwi, Bdago3 and Bdaub. After the observation of the ovary after RNAi, the phenotype of lower ovary diameter was presented, which demonstrated the RNAi to Bdtud caused the delayed development of ovary. Putting the treated female and normal male together for the copulation exprement and the results showed the treated females presented less interestedin copulation. Thus, the amount of eggs in treatment were less than the control’s. Furthermore, the RNAi to Bdtud also caused the lower expression of polar cell formation genes, which suggested that the RNAi may affect the formation of polar cells. The investigation demonstrated that Bdtud could be a potential target of the pest management and effectively control the pest population via RNAi.4. The DGE analysis to tudor RNAiThe DGE analysis was performed to investigate the differential expressed genes after RNAi to Bdtud. The results revealed that 188 and 558 genes were down and up regulated, respectively, based on the standard of log2RPKM≥1. Among the down-regulated genes, the majority were correlated with cell formation process, while the up-regulated genes mainly contained transfactor and enzymes correlated with transcription. The preliminary results revealed that RNAi to Bdtud actually caused the variation of cell differentiation and formation in ovary development.5. The cloning and RNAi of the BdTre2In order to investigate the effect of trehalose metabolism to ovary development, based on the transcriptome, the membrane-bound trehalase gene was cloned by RACE and PCR. The full length of BdTre2 is 2,715 bp, in which the open reading frame,5’ UTR and 3’UTR is 1,842 bp,549 bp and 324 bp, respectively, and encodes 613 aa. The analysis to the sequence showed that this gene have a transmembrane region and 2 trehalase signatures, which demonstrated that this gene belongs to membrane-bound trehalase.The results of RNAi to BdTre2 showed the silence efficiency reached 80%, meaningthat this gene could be effectively silenced. Subsequently, the suppression of the gene mediated the increase of trehalose concentration. According to the described method, the diameter of the treated ovary was significant bigger than control, suggesting that the trehalase may have a negative regulation effect onthe ovary development.In summary, based on the ovary transcriptome of B. dorsalis and qPCR, the expression patterns of 10 pathways, including 30 genes, in different tissues were analyzed. Then the piRNA pathway and trehalase gene were selected for functional analysis. The results showed these genes could affect the growth of ovary and thus affected a series of physiological activities. This investigation provides a scientific basis for the research ongenes related to development of ovary and effective management to B. dorsalis. |
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