Abstract: 【Objective】 The objective of this study is to construct RNAi vector containing p23 gene of Citrus tristeza virus (CTV), and obtain transgenic orange plants with virus resistance. 【Method】 Based on the pathogen-derived resistance and the CTV genome sequences published by NCBI, two specific conserved fragments of p23 with different lengths were cloned. Two segments and vector pBI 121 were double-digested and connected for RNAi construction. Subsequently, to initially estimate the antiviral feasibility, the Mexican lime (CTV indicator plant) leaves were injected with Agrobacterium contained the RNAi vector for transient expression, and observed using GUS histochemical staining method. The leaves were inoculated with CTV T36 isolate, and detected by enzyme-linked immunosorbent assay (ELISA). The RNA of leaves was also extracted and reverse transcript to cDNA. Quantitative real-time PCR (q-PCR) was performed to observe the CTV p20 gene expression which could reflect the virus in hosts. The RNAi vector was also transferred into the epicotyl stem of ‘DA HONG’ sweet orange seedlings via Agrobacterium-mediated transformation. Resistant buds were engrafted onto Carrizo Citrange in vitro seedlings. DNA extracted from the ‘DA HONG’ sweet orange leaves was used as the PCR template to identify the transgenic plants. Plants containing the target gene were re-engrafted onto sour orange seedlings and stored in the greenhouse. The expression of the p23 within the transgenic plants was evaluated by q-PCR. The skin buds of CTV T36 isolate hosts were collected and inoculated onto the transgenic sweet orange. The leaves from the sprouted branch tips were collected and analyzed the pathogen resistance capability with the same method that the transient expression leaves detected. Plants without detectable virus infection after the first inoculation were also inspected and analyzed by the same manner in the second round. 【Result】 Long (513 bp) and short (291 bp) fragments of the p23 were cloned. These p23 fragments are then cloned into the pBI121 vector, named p23-RNAi. This p23-RNAi vector was then delivered in the Mexican lime leaves using the Agrobacterium-mediated transient expression assay. The leaves were identified based on the presence of blue stains after GUS staining, indicating that the Agrobacterium contained vector p23-RNAi may induce transient expression in Mexican lime leaves. On the 15th and 30th day after the CTV inoculation, the ELISA detection results for the transgenic Mexican lime leaves in p23-RNAi plants were all negative, whereas the q-PCR detection results showed that the accumulation level of p20 expression was significantly lower than that of the control plants. It indicated that the transient expression of p23-RNAi may, in a defined period, inhibit the CTV infection. Introduction of p23-RNAi via Agrobacterium-mediated genetic transformation led to the production of resistant buds for the ‘DA HONG’ sweet orange, and PCR amplification confirmed a total of seven transgenic plants. The expression of the p23 in all seven transgenic plants was further confirmed by q-PCR amplification, and the gene expression level exhibited a certain degree of difference, with expression level in plant E being the highest, followed by C, F, H, A, B, and G. After inoculation with CTV, the level of expression of p20 varied among these seven transgenic plants, with plant A having the highest level of p20 expression, followed by G, F, E, B, H and C. The transgenic plants showed higher pathogen resistance, albeit to different degrees, when compared to the control plants. However, the virus resistance degrees in the transgenic plants were not closely related to the expression levels of the exogenous gene. For example, plant E, which had the highest expression level of the exogenous gene, did not exhibit powerful CTV resistance. By contrast, the transgenic plant C displayed complete resistance after the two rounds of virus inoculation. 【Conclusion】The p23-RNAi construct that generated confers plant disease resistance to CTV. Transient expression assay can be applied for the high efficiency identification of resistance and screening for high efficiency RNAi vectors.

Key words: Citrus tristeza virus (CTV), p23, RNAi, ‘DA HONG&rsquo, sweet orange, transient expression, genetic transformation