| RNAi is triggered by ds RNA targeting m RNA causing highly specific cleavage of target gene. Because RNAi is highly efficient and convenient, it is widely used in gene function studies, high-throughput screening of target genes, gene therapy, drug target prediction and other areas, including control of agricultural pests. RNAi can be achieved by microinjection, soaking and feeding method. Although RNAi is a widely present in a variety of eukaryotic and is highly conserved mechanism. Its application still face many problems. For example, in some species it is difficult to achieve RNAi and RNAi effects last very short time. Oriental fruit fly is an important agricultural pest feeding on up to 250 fruits and vegetables. Through the establishment of oriental fruit fly feeding RNAi system, we studied the effects of long-term feeding. We discovered that Bactrocera dorsalis can become refractory to RNAi due to the change of endocytosis function. We also use RNAi technique to study the function of spr gene and symbiotic virus function.1. We established B. dorsalis feeding RNAi system. The results showed that both directly feeding the target ds RNA and feeding E. coli expressing ds RNAs down-regulated target gene expression. In rpl19 ds RNA feeding group, rpl19 expression showed a 90% decrease on the first day and seven day. In the E. coli feeding group, the target gene exihibted a 30% down-regulation on the first day and seven days. In the V-ATP-D RNAi experiments, the target gene expression lowered by about 30%. We also found that both feeding ds RNA and E. coli expessing ds RNAs caused target gene expression up-regulated 2 or 4 days after feeding. Tissue-specific test results show that RNAi caused by feeding is not limited to the intestine, it can be observed in the nervous system, the reproductive system and the fat body. Feeding noa ds RNA caused noa expression dropped 64% 23% 95% and 89% 2 days after feeding in head, ovary, testes and fatbody, respectively Experimental results also showed that after continuous feeding rpl19 ds RNA for 12 days, the target gene expression levels returned to normal, showing no significant difference from the control group. At the same time, this phenomenon is not related to the flies age and ds RNA concentration. Using either 500 ng/μl 100 ng/μl or 10 ng/μl rpl19 resulted in the recovery of target gene expression 12 days after the bioassay. The study using 5 days old and 10 days old flies also showed the same resules. Results also showed that F2 generation showed normal RNAi effect indicating that RNAi interference in the F1 generation has no effect on the progeny. Day 0 flies in the F2 progeny showed 66% down-regulation in the RNAi feeding bioassay, which is the same as that in the untreated group.2. The results showed that the first RNAi targeting endogenous gene can make the flies become refractory to subsequent RNAi, making the secondary RNAi can not successfully reduced the level of gene expression. In the secondary RNAi, as expected, the naive group showed efficient RNAi after secondary exposure to a ds RNA targeting rpl19; in this group, rpl19 expression decreased by 66%e. However, in the challenged group, after secondary RNAi, depletion of rpl19 could not be observed. At the same time this refractoriness is non-gene-specific. The duration of this refractoriness is relevant to the ds RNA concentration used in the first RNAi. The refractoriness caused by 1000 ng/μl lasted for 20-30 days; 10-20 days for 100 ng/μl treatment and no more than 10 days for 10 ng/μl treatment. Bafilomycin treatment showed that ds RNA uptake is mediated by endocytosis mechanism. Fluorescence staining results showed that ds RNA uptake mechanism was repressed in the RNAi refractory flies. ds RNA entry was blocked. Quantitative PCR results showed that key genes associated with endocytosis were significantly reduced. For example, chc light cog3 ap50 and nina c dropped by 30-50%. This conclusion is also supported by high-throughput sequencing results. High-throughput sequencing found that some key genes involved in cell transport were decreased. For instance, chc actin1 actin2 and actin5 all showed down-regulation. This conclusion is further supported by digital gene expression analysis in first RNAi exposure using 10ng/μl rpl19 ds RNA. These results indicate that the inability of a low ds RNA concentration 10ng/μl to induce RNAi is due to the disruption of the endocytic pathway.3. We cloned the full sequence of spr gene. This gene encodes a 324 amino acids, which is very conservative, with the similarity 86% E = 0 compared to Mediterranean fruit fly. Expression profiles showed that spr showed highest expression in the midgut, hindgut and head. spr also expressed at different developmental stages, among which the highest levels of expression is in the larval and pupal stages. The results show that the survival rate of female flies decreased significantly in the H2O2 bioassay after mating, The Survival rate of mated flies is 9.4% 72 h after the bioassay. Meanwhile, the survival rate of unmated flies is 56.7% p=0.017 And the genes in the target of rapamycin TOR signaling pathway were up-regulated, suggesting that a successful mating decreases the resistance to oxidative stress in female flies. For example, rheb up-regulated by 88% and 28% for tor gene The key factor gene s6 k was up-regulated by 54%. But the resistance to oxidative stress remained unchanged in mated female flies after knocking down Bd SPR expression by 75% by RNAi technique. These studies demonstrated that Bd SPR mediates B. dorsalis post-mating change of sensitivity to oxidative stress through TOR pathway.4. Through the DGE and transcriptome sequencing, we identified eight fruit fly symbiotic viral sequences. contig1697, contig475, comp2791 and comp11365 encoding structural proteins or non-structural proteins of bicistronic virus family virus. comp3227 and comp16524 encoding Rd Rp Polymerase belonging Rhabdoviridae family. We analyzed comp11365 virus Rd Rp sequence and confirmed it is a small RNA virus classified as Picornavirales, bicistronic virus family, cricket paralysis virus genus, and named it Bdv-1. Bdv-1 virus is mainly distributed in the fly gut, which is 3,000 times higher than that in the reproductive system. We used the RNAi technology to remove the virus and found that total gut bacteria decreased. After knocking out the virus, the total intestinal bacteria dropped by 48%. γProteobacteria, Bacteroidetesa and Actinobacteri all showed a 50% decrease after Bdv-1 RNAi. αProteobacteria and Firmicutes did not show significantly difference between Bdv-1 RNAi and its control. !... |
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