| Tristeza, which is caused by Citrus tristeza virus (CTV), is one of the most economically citrus diseases and has caused huge economic losses to citrus production worldwidely. CTV, a member of the Closterovirus genus, has a single-stranded, positive-sense genomic RNA (+ssRNA). The CTV genome is about20kb long, organizing12open reading frames (ORFs) which encode19proteins. CTV is transmitted by the brown citrus aphid in a semi-persistent manner. In this study, prokaryotic expression vectors of pET28a and pET22b for CTV p20and p23was constructed respectively, and p20and p23protein were expressed. The quantity of p25protein in CTV different isolates was detected by the application of enzyme-linked immunosorbent assay with its polyclonal antibody. The concentration differences of virus particles were evaluated by SYBR Green I RT-qPCR. The numbers of aphids and the transmission rates were analyzed by feeding aphids with protein p20, p25and p27through an artificial membrane. This study provided a theoretical base for CTV epidemiology, mechanisms of aphid transmission and aphids-CTV-host interaction research.The main results were as follows:1. A set of primers were designed within the construct of pET28a and pET22b and gene p20and p23, and gene cloning and DNA sequencing were performed. The expected clone was transformed into BL21(DE3) and proteins were expressed by IPTG induction.2. The quantity of p25protein of different CTV isolates was detected by ELISA with p25polyclonal antibody. OD405of leaf samples from CTV isolates (CT11A, CT14A, CT16-2, CT77and CTlijian) were2.7246,1.6607,1.1093,0.8508and0.8366, respectively. The result indicated that the content of p25protein decreased in CT11A, CT14A, CT16-2,CT77and CTlijian.3. The content of virus particles was detected by SYBR Green I RT-qPCR. The results indicated the content of CTV isolates decreased in CT11A, CT14A and CT16-2. However, The quantity of virion from CTV isolates was not completely consistent with aphids transmission rate, suggesting that virus concentration could influence transmission rate and other factors also could be involved in CTV transmission by aphids.4. Recombinant protein p25, p27and p20were added to aphid feed. Then aphids were transferred to CT11A, CT16-2and CTlijian. RT-qPCR was used to analyze CTV concentrations in aphids. Compared with the control, CTV concentrations in aphids obtained from CT11A increased after feeding p27protein and CTV concentrations in other aphids reduced.5. Recombinant protein p25and p27were added to aphid feed. Transmission rate of5aphids in CTlijian were31.03%and35.71%, respectively. Transmission rate of5aphids which only ate feed was40%. |
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