| The 2b gene of cucumber mosaic virus (CMV) is an important virulence gene in CMV genome, and 2b protein is the suppressor of the post-transcriptional gene silencing (PTGS) in plant. Virus gene mediated strategy has been widely adopted to improve virus resistance in crops. However, no case of applying CMV 2b gene to obtaining virus resistance has been reported. Based on the latest understanding of PTGS, we tested 2b gene derived virus resistance to CMV. We developed a novel one-step RT-PCR approach, which avoided the extraction of the virus, and attempted to amplify 2b gene fragments from 9 isolates collected in China. The full-length 2b gene of CMV-BG was selected to construct a PVX infectious vector to analysis the interaction between sense and antisense 2b gene. Furthermore, both sense and antisense 2b gene of CMV-BG were introduced into Nicotiana tabaccum var. SR1 and Lycopersicon escuentum var. Hongmanao 213 respectively by Agarobacterium transformation. Resistance evaluation of the transgenic tobacco plants revealed 2 transgenic plants showing high-level resistance to CMV-BG. The main results in this thesis are as follows:1 Using a Chinese CMV isolates originated from tomato in Beijing as template, and primers designed from the conservative regions of reported CMV sequences, a novel one-step RT-PCR system was developed. The advantages of the system are simplified sample preparation by grinding a leaf-disc directly in virus extraction buffer and eliminating complicated virus RNA extraction. Sensitivity comparison between the novel one-step RT-PCR and DAS-ELISA for PVX revealed that the former method was 20 times more sensitive than the later one. The novel one-step RT-PCR system was also successfully tested with field samples of turnip mosaic virus (TuMV) in 8 different crucifer vegetable crops. The results suggested that the novel method was suitable for detecting virus rapidly without complicated extraction of virus RNA, which provides a great advantage when detecting large number of samples in the same time.2 Using the one-step RT-PCR method, we amplified successfully 2b gene fragments with a length of 300bp covering 90% of 2b gene from 9 different CMV isolates collected from vegetable crops in China. All these fragments were introduced into pMD18-T vector, and 7 of them were sequenced. The sequence comparison suggested that 2b gene fragments of different Chinese CMV isolates were highly conservative, and the identity of nucleotide and deduced amino acid sequences were over 93% and 90%, respectively. It showed that all the analyzed isolates belong to CMV subgroup I. High homology of 2b gene among different CMV isolates suggested that resistance derived from 2b gene of one isolate might provide resistance to the other isolates.3 Using PVX-expression vector (pSfinx), quick analyzing of the interaction between sense and antisense 2b gene of CMV-BG was performed before introducing it into acceptor plants. Firstly, the full-length 2b gene of CMV-BG was amplified by one-step RT-PCR and inserted into pSfinx to obtain the PVX-based infectious vector pVX2bS and pVX2bAS in Agrobacterium tumefaciens strain MOGIOI containing the sense and antisense 2b gene, respectively. Both pVX2bS and pVX2bAS were able to infect N. benthamiana, N. tabacum var. SRI, N. tabacum var. Samsun NN, Solanum tuberosum var. Shepody and Lycopersicon escuentum var. zhongshu No. 5 as well as pSfinx, but showed disease symptom delayed effect comparing to pSfinx. The two vectors could hold integrated 2b gene in leaves of N. tabacum var. SRI over 45 days under 22癈/16癈 (day/night temperature). Interaction between sense and antisense 2b gene was analyzed by inoculating on SRI with mixture of two A. tumefaciens with each of the vectors, or by cross-inoculation. The results indicated that presentation of only one vector (pVX2bS or pVX2bAS) could be identified randomly 15 days post-inoculat...
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