中国农业科学 ›› 2022, Vol. 55 ›› Issue (24): 4840-4850.doi: 10.3864/j.issn.0578-1752.2022.24.005

• 植物保护 • 上一篇    下一篇

柑橘黄化花叶病毒侵染性克隆构建及应用

蒋琪琪(),许建建(),苏越,张琦,曹鹏,宋晨虎,李中安(),宋震()   

  1. 西南大学柑桔研究所/国家柑桔工程技术研究中心,重庆 400712
  • 收稿日期:2022-07-18 接受日期:2022-09-13 出版日期:2022-12-16 发布日期:2023-01-04
  • 通讯作者: 李中安,宋震
  • 作者简介:蒋琪琪,E-mail:j13206107876@163.com。|许建建,E-mail:swuxujj@foxmail.com
  • 基金资助:
    国家重点研发计划(2021YFD1400800);重庆市自然科学基金(CSTB2022NSCQ-MSX0752)

Construction and Application of Infectious Clone of Citrus Yellow Mosaic Virus

JIANG QiQi(),XU JianJian(),SU Yue,ZHANG Qi,CAO Peng,SONG ChenHu,LI ZhongAn(),SONG Zhen()   

  1. Citrus Research Institute, Southwest University/National Citrus Engineering Research Center, Chongqing 400712
  • Received:2022-07-18 Accepted:2022-09-13 Online:2022-12-16 Published:2023-01-04
  • Contact: ZhongAn LI,Zhen SONG

摘要:

【目的】构建柑橘黄化花叶病毒(citrus yellow mosaic virus,CYMV)侵染性克隆,为深入研究其分子特性及致病机理打下基础。【方法】利用In-Fusion同源重组技术将分段扩增的CYMV基因组序列与三元表达载体pCY重组连接,构建该病毒1.4倍基因组全长DNA克隆并开展序列分析。将所获克隆通过农杆菌介导接种尤力克柠檬实生苗,通过分子检测、症状和病毒粒子观察及再嫁接实验鉴定其侵染性。利用所获侵染性克隆接种不同的柑橘品种及草本植物,通过RT-PCR检测确定侵染率,观察不同柑橘品种受侵染后的症状差异。基于侵染性克隆构建ORFⅠ和ORFⅡ分别替换为绿色荧光蛋白基因(green fluorescent protein,gfp)的突变体,分析突变对病毒侵染性的影响。【结果】利用In-Fusion同源重组技术获得CYMV 1.4倍基因组全长DNA克隆7个。其中,CYMV-3与已登录GenBank的9个CYMV分离株基因组相应核苷酸序列一致性为90%—100%,与分离株CYMV-SO(AF347695)同源性最高并在遗传进化树上聚为一簇。侵染性鉴定结果表明,CYMV-3接种的14株尤力克柠檬植株中有4株呈RT-PCR阳性,并观察到点状褪绿症状;提取侵染植株系统新叶DNA,PCR扩增获得预期的覆盖CYMV基因组全长的特异性片段;以CYMV-3侵染显症植株为毒源嫁接接种粗柠檬实生苗,通过RT-PCR在90 dpi可检测到CYMV特异性条带;取CYMV-3侵染显症植株的新叶进行固定切片并电镜观察,可见大小约130 nm×30 nm的杆状病毒粒子,说明CYMV-3为CYMV侵染性克隆。将CYMV-3通过农杆菌介导真空浸润接种10个柑橘品种,注射接种5个柑橘品种并进行RT-PCR检测,结果显示前者侵染率为11.11%—100.00%,后者侵染率为63.16%—90.00%,部分柑橘品种出现黄化、花叶、斑块状黄化等CYMV侵染症状。通过农杆菌介导注射接种本氏烟、玉米、豇豆和高粱,结果显示仅高粱检测到RT-PCR阳性植株,且系统叶片沿叶脉周边出现条状褪绿带,表明CYMV可以侵染高粱。构建了CYMV-3 ORFⅠ和ORFⅡ分别替换为gfp的突变体,通过农杆菌介导注射接种粗柠檬,RT-PCR检测结果均为阴性,且无侵染症状。【结论】成功构建了CYMV的侵染性克隆,通过农杆菌介导注射可高效接种柑橘植株,侵染症状在不同品种上具有差异。

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