关键词: 柑橘黄化花叶病毒, 侵染性克隆, 农杆菌介导接种, 突变体

Abstract:

【Objective】An infectious clone of citrus yellow mosaic virus (CYMV) was constructed to lay a foundation for further study of its molecular characteristics and pathogenic mechanism. 【Method】The amplified CYMV genomic sequence segments were recombined with the ternary expression vector pCY using In-Fusion homologous recombination technology, and a 1.4-fold full-length genomic DNA clone of the virus was constructed and sequenced. The obtained clones were inoculated onto seedlings of Eureka lemon by Agrobacterium-mediated vacuum infiltration (AVI), and their infectivity was identified by molecular detection, presentation of symptoms and virus particles, and re-grafting determination. The obtained infectious clones were used to inoculate different citrus varieties and herbs, and the infection rate was determined by RT-PCR and the various symptoms of different citrus varieties after infection were observed. Mutants of the infectious clones with ORFⅠand ORFⅡ replaced by green fluorescent protein gene (gfp), respectively were constructed, and subjected to infectivity analysis. 【Result】Seven clones of 1.4-fold CYMV genome-length DNA were obtained by In-Fusion homologous recombination technology. Among them, CYMV-3 was 90%-100% identical to the corresponding genomic nucleotide sequences of nine CYMV isolates registered in GenBank, and had the highest homology with CYMV-SO isolate (AF347695) and was clustered on the same branch of the genetic evolutionary tree. The results of infectivity identification showed that four out of the 14 Eureka lemon plants inoculated with CYMV-3 were positive by RT-PCR detection, and punctate chlorotic symptoms were observed. DNA was extracted from the new systemic leaves of the infected plant and the expected specific fragments covering the full length of the CYMV genome were obtained by PCR amplification. CYMV-3 infected plants with symptoms were used as the virus source to graft and inoculate rough lemon seedlings, and CYMV specific bands could be detected by RT-PCR at 90 dpi. New leaves of CYMV-3 infected plants with symptoms were taken for fixation and sectioning for electron microscopy and baculovirus particles of about 130 nm × 30 nm were observed, indicating that CYMV-3 was a CYMV infectious clone. Ten and five citrus varieties were inoculated by CYMV-3 via Agrobacterium-mediated vacuum infiltration and injection, respectively, and were tested by RT-PCR. The results showed that the infection rate of the former was 11.11%-100.00% and the latter was 63.16%-90.00%. Some citrus varieties appeared CYMV symptoms such as yellowing, mosaic and yellowing patches. Nicotiana benthamiana, Zea mays, Vigna unguiculata and Sorghum bicolor were inoculated by CYMV-3 via Agrobacterium -mediated injection, and only S. bicolor plants were detected to be positive in RT-PCR detection, and chlorotic strips appeared along the veins of the systemic leaves, indicating that CYMV can infect S. bicolor. The mutants of CYMV-3 with ORFⅠand ORFⅡ replaced by gfp, respectively, were constructed and inoculated onto rough lemons by Agrobacterium-mediated injection. All the inoculated plants were negative by RT-PCR detection and showed no obvious symptoms. 【Conclusion】The infectious clone of CYMV was successfully constructed and could infected citrus plants efficiently via Agrobacterium-mediated injection, resulting different symptoms in different varieties.

Key words: citrus yellow mosaic virus (CYMV), infectious clone, Agrobacterium-mediated inoculation, mutant