| Southern rice black-streaked dwarf virus(SRBSDV), transmitted by Sogatella furcifera(Horváth), is a great threat to rice production in East Asia. To elucidate the interaction between WBPH and SRBSDV,We sequenced the salivary gland transcriptomes of viruliferous(SRBSDV-infected) and nonviruliferous adult WBPHs using the Ion Proton II, PGM platform; We cloned and analyzed 9 reference genes of WBPH under different experimental conditions; an absolute quantification qPCR method was utilized to detect viral gene expression levels at different developmental stages of WBPHs fed on SRBSDV-infected rice plants.1. The salivary glands of the WBPH play an important role in the feeding behaviour and transmission of viruses. We sequenced the salivary gland transcriptomes of viruliferous(SRBSDV-infected) and nonviruliferous adult WBPHs using the Ion Proton II, PGM platform. A total of 53,073 unigenes were obtained, with 51,407 unigenes from nonviruliferous salivary glands(NVSGs) and 52,062 unigenes from viruliferous salivary glands(VSGs). The GO annotation and KEGG pathway analyses indicated that many genes related to metabolism were significantly upregulated. According to annotations against the nr database, many of the unigenes identified were associated with the most studied enzymes in hemipteran saliva. A total of 579 potential secretory proteins were predicted, some of which might be involved in virus-vector interactions. A comparative analysis of the transcriptomes of the NVSGs and VSGs found that 89 genes had a significantly different level of expression, which indicated that these unigenes might be associated with virulence traits. Of these genes, the expression profiles of vitellogenin(Vg) and odorant-binding protein(OBP) were determined for different tissues(i.e., salivary glands, gut and remaining bodies) from male and female, nonviruliferous and viruliferous adult WBPHs.2. Here we cloned and sequenced partial fragments of 9 commonly used housekeeping genes(including elongation factor 1-alpha(EF1A), Polyubiquitin(UB), ribosomal protein S18(RPS18), actin1(ACT), alpha-1-tubulin(A1TU), glyceraldehyde-3-phosphate(GAPDH), ribosomal protein L9(RPL9), ribosomal protein L10(RPL10) and 18 S RNA(18S) of S. furcifera, and their stability under different experimental conditions(developmental stage, temperature stress, body part and acquisition of a plant virus) were also checked. Expression stability of nine selected reference gene were evaluated by four freely and commonly used software tools, geNorm version3.5, NormFinder version 0.953, BestKeeper, and the delta Ct method. Based on the results of RefFinder, A1 TU, RPL9, GAPDH and A1 TU were found to be the most suitable reference genes in different developmental stages, acquisition of plant virus, body parts and temperature stress.3. An absolute quantification qPCR method was utilized to detect viral gene mRNA expression levels at different developmental stages of WBPHs fed on SRBSDV-infected rice plants. A comparison of viral copy numbers of the SRBSDV S10 gene at the same developmental stage indicated that the WBPH had higher viral copy numbers when the virus was acquired at the earlier developmental stages. The adult stage WBPHs that had acquired the virus at the 1st-2nd nymphal stage displayed significantly higher viral titers than WBPHs that acquired the virus at the 3rd-4th nymphal stage, at the 5th nymphal stage, and at the adult stage. The 5th nymphal stage WBPHs that acquired the virus at the 1st-2nd nymphal stage displayed higher viral copy numbers than 5th nymphal stage WBPHs that acquired the virus at the 3rd-4th nymphal stage and at the 5th nymphal stage. The highest viral load value appeared in the middle adult stage. The annual immigration characteristics of WBPHs would be beneficial for the dispersal this virus. |
