Guangdong nongye kexue (Aug 2023)
Construction and Identification of Infectious Clone from Sri Lankan Cassava Mosaic Virus Tvm3 Isolate
Abstract
【Objective】Sri Lankan cassava mosaic virus (SLCMV) is a circular single-stranded DNA virus, containing two components of DNA-A and DNA-B. SLCMV is a member of the genus Begomovirus in the family Geminiviridae, which is a persistent threat to the world's cassava industry. In 2018, the SLCMV was found and reported for the first time in cassava from Fujian and Hainan provinces, China. However, the viral resistance of cassava varieties in domestic was unknown and the germplasm resources for disease resistance was lacking. To further explore the genetic background of domestic cassava germplasm resources resistance to mosaic disease and the mechanism of interaction between virus and host, SLCMV infectious clone was constructed and verified.【Method】In this study, SLCMV TVM3 strain was used as the object, and its 1.10 mer DNA-A and 1.08 mer DNA-B components were respectively constructed into pCAMBIA1301 vector, and the viral infectious clone vectors pCAMBIA1301-DNA-A (pDNA-A) and pCAMBIA1301-DNA-B (pDNA-B) were obtained. Agrobacterium tumefacien GV3101 mediated pDNA-A and pDNA-B co-infected tobacco and Arabidopsis plants.【Result】At 14 d post inoculation, the systematic leaves of tobacco plants produced severe mosaic and distortion symptoms, while the systematic leaves of Arabidopsis plants showed mild distortion at 18 d post inoculation. Total DNA was extracted from SLCMV infected tobacco and Arabidopsis plants leaves, and viral DNA-A and DNA-B components were detected by PCR method. In addition, the virus infection rate of tobacco plants was 100%, while the virus infection rate of Arabidopsis plants was only 40%, and the difference between the two group was significant (P < 0.05).【Conclusion】The SLCMV infectious clone have been successfully constructed, which could infect tobacco and Arabidopsis plants, and the viral infection rate reached 96.67% in tobacco plants. Furthmore, the DNA sequences of infectious clone in pDNA-A and pDNA-B are 1.10 mer and 1.08 mer length of the original virus genomic DNA-A and DNA-B components, respectively, whichdid not contain repetitive coding region sequences. This study provides an important basis for the further explore the genetic background of domestic cassava germplasm resources resistance to mosaic disease and the pathogenesis of virus.
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