Studies on Tobacco mild green mosaic virus and construction of a TMGMV-based vector
Date Issued
2011
Date
2011
Author(s)
Yao, Jun-Yi
Abstract
Peppers, belonging to the genus Capsicum in the family Solanaceae, originated from South America and now become important crops worldwide. According to the literatures, there are at least 8 tobamovirus species infecting peppers. TMV and PMMoV have been isolated from the field in Taiwan. In addition, our lab first reported a pepper disease caused by TMGMV in 2003. To understand the disease incidence caused by TMGMV, the degenerate primers for pepper-infecting tobamoviruses were designed for preliminary detection. After that, we separately designed specific primers for TMGMV and two frequently found viruses in pepper (TMV and PMMoV). A multiplex RT-PCR was developed using the specific primers for these three viruses and plant mitochondria nad5 gene. Pepper samples collected from Ilan, Hualian, Taipei, Taoyuan and Taichung areas were first detected by tobamovirus degenerate primers. Four out of 108 samples were infected by tobamovirus. According to the following multiplex RT-PCR, the target viruses were not detected in these 4 samples. This result suggested that these samples may be infected by other tobamovirus. Although we did not detect TMGMV in the survey, the tobamovirus specific primers and multiplex RT-PCR can still be used in the future. Not only the occurrence of TMV and PMMoV can be investigated, but also the infection of TMGMV in peppers can be confirmed by this detection method. Viral vectors have been constructed for medical application, identifying unknown gene function and studying the interaction between virus and its host. Since TMGMV has never been reported as a viral vector, we used two different ways for vector construction. One (pCRII-TG2) is to delete most of the CP gene, and the other (pCRII-TG3) is to insert an additional CP subgenomic promoter (sgp). The EGFP gene was inserted into the viral vectors to construct pCRII-TG2E and pCRII-TG3E. RNA transcripts of TMGMV clones (p5-6) and its derived vectors were inoculated to tobacco protoplasts and plants. The results showed that TMGMV vectors could accumulate their RNAs and expressed green fluorescence in tobacco protoplasts. pCRII-TG3 could move systemically and also expressed CP in plant. On the contrary, the RNA accumulation of pCRII-TG2E and pCRII-TG3E carrying EGFP gene were lower than pCRII-TG2 and pCRII-TG3 in protoplasts, and the symptoms induced by pCRII-TG2E and pCRII-TG3E were delayed in local lesion host. The green fluorescence signal could be observed around the local lesions only in the plant inoculated by pCRII-TG3E. The RNA accumulation in the inoculated leaves of pCRII-TG2E and pCRII-TG3E were lower than that of pCRII-TG2 and pCRII-TG3 and green fluorescence failed to be observed in systemic host plant. These results indicated pCRII-TG3E lost its ability of systemic movement in this host. In the future, we have to improve the TMGMV-based vectors in order to express foreign gene in planta and to use them in studying the interaction between TMGMV and host or other viruses.
Subjects
Capsicum spp.
Tobamovirus
TMGMV
viral vector
Type
thesis
File(s)![Thumbnail Image]()
Loading...
Name
ntu-100-R97633017-1.pdf
Size
23.54 KB
Format
Adobe PDF
Checksum
(MD5):aab0c863b716756b180e7ca99e72c5de