Abstract:

【Objective】The objective of this study is to construct infectious clone of citrus vein enation virus (CVEV), and to lay a foundation for analyzing its pathogenic mechanism at the molecular level. 【Method】5′ RACE was performed using SMARTer^?RACE Kit to confirm the exact 5′ sequences of CVEV. A specific primer pairs for amplification of CVEV genome-length cDNA was designed according to the sequence analysis result and the conservative sequence of isolate VE-1. Genome cDNA was amplified with EV25-F/EV5983-R primers using total RNA of CVEV infected fragment. The full-length cDNA of CVEV and linearized pXT1 were connected by In-Fusion recombination. Genome-length cDNA clones of CVEV were identified by PCR and sequencing analysis, and then subjected to agrobacterium-mediated inoculation. Citrus aurantium, C. paradisi, C. limon, C. paradisi × Poncirus trifoliata, P. trifoliata × C. sinensis, P. trifoliata were inoculated by agrobacterium-mediated vacuum infiltration. The infectivity of the cDNA clone was further identified by RT-PCR detection and symptom observation. 【Result】An RT-PCR amplification system for full-length genome of CVEV was established, and 10 full-length cDNA clones of CVEV genome based on the pXT1 were obtained. Six clones, namely CVEV1901-CVEV1906, were randomly selected and sequenced. The nucleotide sequence identity of them was 99.35%. Among them, the CVEV1901 is 5 983 nt in length and consists of five open reading frames (ORF), two untranslated regions (UTR) at the 5′ end (207 nt) and 3′ end (198 nt), and an intergenic region (IR) of 122 nt between ORF2 and ORF3. The nucleotide sequence analysis results showed that the identity of CVEV1901 and CVEV isolates from China was the highest, and that of Zhejiang isolate XZG and Sichuan isolate SM was 99.98% and 99.11%, respectively. The nucleotide sequence identity of CVEV1901 and CVEV isolate VE-1 from Spain, VE701 from California, America and IBK from Japan was 96.89%-98.61%. CVEV1901 showed much lower nucleotide sequence identity with pea enation mosaic virus and alfalfa enamovirus (about 90%) in the same genus. CVEV1901 was inoculated onto 6 different citrus varieties by agrobacterium-mediated vacuum infiltration. RT-PCR detection results at 120 days after inoculation showed that the positive plant/inoculated plant (positive rate) of C. aurantium, C. paradisi, C. limon, C. paradisi × P. trifoliata, P. trifoliata × C. sinensis, P. trifoliata was 16/17 (94.12%), 12/14 (85.71%), 16/21 (76.19%), 15/19 (78.95%), 13/14 (92.86%) and 0/18 (0), respectively. Among them, some C. aurantium showed typical symptoms of CVEV infection, with small auricular-shaped protrusions in the lateral veins of the leaves, and corresponding depressions in the back of the leaves. Some C. paradisi and C. limon had shrunken leaves. 【Conclusion】An RT-PCR amplification system for the full-length CVEV genome was established, and an infectious full-length cDNA clone of CVEV was obtained. The full-length cDNA clone can induce typical CVEV infection symptoms on C. aurantium, C. paradisi, C. limon by agrobacterium-mediated vacuum infiltration.

Key words: citrus vein enation virus (CVEV), infectious clone, genome-length RT-PCR