Construction Of RNAi Vectors Of Citrus Tristeza Virus, Genetic Transformation And Evaluation Of Transgenic Plant

Citrus industry is an important pillar industry in South China areas. However, the industry has been threatened by Citrus tristeza virus (CTV), a positive-sense single-stranded RNA virus. Since many examples of using RNAi to control plant RNA viruses have been reported, it is possible to RNAi to control CTV in citrus.When plant RNA viruses infect plants, in order to survive, they usually inhibit host's RNAi mechanism by expressing gene-silencing suppressor genes. Our rationale is that we would be able to fight against plant viruses by inactivating the expression of their gene-silencing suppressors. In this study, three suppressor factor genes p25, p20 and p23 of CTV were targeted by RNA interference.(1) Construction of RNAi vectors, transformation and verification of transgenic plantsPCR primers were designed according to the conserved sequences of CTV genomic RNA that encode the three suppressor genes p25, p20 and p23, and RT-PCR was performed to amplify the target sequences. Sequence overlap extension (SOE) method was used to join the amplified products to form inverted repeats. In this way, two multi-gene fragments P2520 and P252023 were obtained.To facilitate the following cloning steps, restriction site Bglâ…¡was added to the down stream sense primer, and Kpnâ… and Bglâ…¡restriction sites were also added to the two nested primers, the upstream primer and the downstream primer, respectively. After TA cloning, the pTZ57R-HP2520 construct, in which HP2520 represents the inverted repeat fragment sequences of CTV's p25 and p20 genes, was obtained. In similar way, the multi-gene construct containing the three suppressor genes was obtained.The pTZ57R-HP2520 and pCAMBIA-2301G were both digested by Sacâ… and BamHâ… restriction enzymes. The purified HP2520 and pCAMBIA-2301G were then ligated and transformed into E. coli DH5a. The transformation was verified by double digestion of the plasmid with Sacâ… and BamHâ… extracted from the transformed cells. The plasmid was then transferred into Agrobacterium strain LBA4404.Transgenic plants were obtained by co-culturing the epicotyls of a sweet orange cultivar Jingcheng with the pCAMBIA2301-HP2520-containing Agrobacterium. Consequently,20 kanamicin resistant adventitious buds were regenerated and 7 of them survived the following grafting operation. PCR was performed using GUS and NPTâ…¡gene-specific primers to detect the transgenes in the transgenic plants. The results showed that six plants were GUS gene positive while all 7 were NPTâ…¡gene positive.The RNAi binary expression plasmid vector pCAMBIA2301-HP252023 was also obtained by same method. Transgenic plants were obtained but only 1 survived after grafting. PCR result demonstrated that the transgenic plant contained both GUS and NPTâ…¡genes.(2) Evaluation of pCAMBIA2301-HP20 transgenic sour orange plants:Seven transgenic sour orange plants containg pCAMBIA2301-HP20, which were obtained previously in the lab, were evaluated. First, GUS staining and PCR amplification of p20 fragment were performed to validate the transgenes. The results showed all of them are true transgenic plants. Second, wild type sour orange plants (as control) and five transgenic plants were inoculated with TRL514, a CTV severe strain, to test and compare their anti-CTV activity. CTV virus RNA detection was performed by RT-PCR 30d,50d and 60d after inoculation. The results showed that CTV RNA was found in control and two transgenic plants but not in the rest three transgenic plants. Therefore, the three CTV RNA undetected transgenic plants may exhibit anti-CTV activity. Further inoculation and study are needed to verify their anti-CTV activity.