柑橘脉突病毒基因组全长cDNA克隆及其侵染性鉴定

【目的】构建柑橘脉突病毒（citrus vein enation virus,CVEV）侵染性克隆,为从分子水平解析其致病机理打下基础。【方法】利用SMARTer^? RACE(rapid amplification of cDNA ends)试剂盒对CVEV的5′序列进行RACE,并依据序列分析结果及CVEV分离株VE-1保守序列,设计CVEV基因组全长cDNA扩增引物。以CVEV毒源植株的总RNA为模板,通过EV25-F/EV5983-R引物扩增CVEV基因组全长cDNA。利用In-Fusion重组连接线性化pXT1和CVEV全长cDNA。通过菌液PCR及测序分析鉴定CVEV基因组全长cDNA克隆。通过农杆菌介导的真空浸润接种摩洛哥酸橙（Citrus aurantium）、邓肯葡萄柚（C. paradisi）、尤力克柠檬（C.limon）、枳柚（C. paradisi×Poncirus trifoliata)、Rusk枳橙（P. trifoliata×C. sinensis）、枣阳小叶枳(P. trifoliata),进一步通过RT-PCR检测、症状观察鉴定所构建CVEV全长cDNA克隆的侵染性。【结果】建立了CVEV的基因组全长RT-PCR扩增体系,获得基于双元载体pXT1的CVEV基因组全长cDNA克隆10个。随机选取的6个全长cDNA克隆CVEV1901—CVEV1906的序列一致性为99.35%。其中,CVEV1901基因组全长5 983 nt,由5个开放阅读框、5′端207 nt和3′端198 nt的两个非翻译区、以及ORF2和ORF3之间122 nt的基因间隔区组成。序列分析结果显示,CVEV1901与浙江分离株XZG及四川SM分离株的序列一致性分别为99.98%和99.11%;与西班牙VE-1分离株、美国加州VE701分离株和日本IBK分离株基因组序列一致性在96.89%—98.61%;与同属中豌豆耳突花叶病毒（pea enation mosaic virus）和紫花苜蓿耳突病毒（alfalfa enamovirus）的序列一致性约90%。通过农杆菌介导的真空浸润将CVEV1901接种至6个不同的柑橘品种,接种后120 d的RT-PCR检测结果表明摩洛哥酸橙、邓肯葡萄柚、尤力克柠檬、枳柚、Rusk枳橙和枣阳小叶枳阳性植株/接种植株（阳性率）分别为16/17（94.12%）、12/14（85.71%）、16/21（76.19%）、15/19（78.95%）、13/14（92.86%）和0/18（0）。其中,部分摩洛哥酸橙出现典型CVEV侵染症状,叶片侧脉和支脉产生耳状小突起,叶背有相应的凹陷;部分邓肯葡萄柚和尤力克柠檬出现叶片皱缩现象。【结论】建立了CVEV的基因组全长RT-PCR扩增体系,获得了CVEV基因组全长cDNA侵染性克隆,通过农杆菌介导的真空浸润接种可引起摩洛哥酸橙、邓肯葡萄柚和尤力克柠檬的CVEV侵染症状。

Construction of Genome-Length cDNA of Citrus Vein Enation Virus and Identification of Its Infectivity

【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.