中国农业科学2025，Vol.58Issue(17)：3451-3460,10.DOI:10.3864/j.issn.0578-1752.2025.17.007
柑橘鳞皮病毒基因组全长cDNA克隆构建及其侵染性鉴定
Construction and Infectivity Identification of Genome-Length cDNA of Citrus Psorosis Virus
摘要
Abstract
[Objective]Citrus psorosis virus(CPsV)is a tripartite,negative-sense single-stranded RNA virus of the genus Ophiovirus in the family Aspiviridae,which can cause cracking of citrus trunks and even the death of the whole plant,seriously threatening the safety of the citrus industry.The construction of reverse genetics systems for negative-strand RNA viruses is challenging.This study aims to establish a full-length cDNA clone of CPsV genome and determine its infectivity,with the expectation of laying a foundation for research on its pathogenic mechanism and other aspects.[Method]The primers were designed using software Primer 5,and the total nucleic acid of CPsV-infected plants was used as a template for RT-PCR amplification of the three strands of CPsV,RNA1,RNA2 and RNA3,respectively.Based on the dual-expression vector pXT1,cDNA clones of three RNA strands were constructed by In-Fusion homologous recombination technology,which were verified by enzyme digestion and sequencing analysis.The cDNA clones of the three strands of CPsV were screened by the Nicotiana benthamiana inoculation system,and further inoculated into the herb host Gomphrena globosa by Agrobacterium-mediated injection,and then inoculated into different citrus varieties by vacuum infiltration to observe their symptoms and perform molecular detection.[Result]A total of two CPsV RNA1 full-length cDNA clones,two RNA2 full-length cDNA clones,and two RNA3 full-length cDNA clones were obtained,respectively.One full-length cDNA clone of RNA1,RNA2 and RNA3 was randomly selected and combined as a full-length cDNA clone of CPsV genome,eight RNA1,RNA2 and RNA3 cDNA clones were selected and inoculated on N.benthamiana through Agrobacterium-mediated inoculation and RT-PCR detection.Among the eight combinations,the positive rate of CPsV-122 was the highest(62.50％).Sequence analysis showed that CPsV-122 had the highest sequence identity with Spanish isolate P-121,and the corresponding sequence identities of RNA1,RNA2 and RNA3 were 98.06％,97.10％and 99.32％,respectively.In a phylogenetic tree based on the amino acid sequence of coat protein,CPsV-122 and P-121 were clustered in the same clade and five isolates from China,Tunisia and Italy.CPsV-122 was inoculated by Agrobacterium-mediated inoculation to G.globosa,Citrus limon and C.paradise for symptom observation and RT-PCR test.The results showed that at 7 dpi,the positive rate of G.globosa was 16.67％(2/12),and at 25 dpi,the positive plants showed obvious reddish-brown blight,local necrosis of leaves and other CPsV infection symptoms.The RT-PCR results of C.paradise and C.limon were negative except for the positive control,but at 90 dpi,13 out of 20 C.limon plants inoculated with CPsV-122 showed obvious CPsV infection symptoms such as dwarfing,yellowing,wilting shoots and shoot gelatinization,while no specific symptoms were observed in the empty vector group and the healthy control group.[Conclusion]CPsV-122 is a full-length cDNA clone of the CPsV genome,which can systematically infect G.globosa and cause typical CPsV infection symptoms on citrus,such as dwarfing and withering of shoots.关键词
柑橘鳞皮病毒/全长cDNA克隆/农杆菌介导接种/侵染性鉴定/柑橘病毒Key words
citrus psorosis virus(CPsV)/full-length cDNA clone/Agrobacterium-mediated inoculation/infectivity identification/citrus virus引用本文复制引用
李娅毓,王新亮,周金环,李楚欣,李佳欣,田旭斌,宋震..柑橘鳞皮病毒基因组全长cDNA克隆构建及其侵染性鉴定[J].中国农业科学,2025,58(17):3451-3460,10.基金项目
重庆市技术创新与应用发展重点项目(CSTB2024TIAD-KPX0063)、国家现代农业(柑橘)产业技术体系(CARS-26-05B) （CSTB2024TIAD-KPX0063）
