Genetic Transformation Of Inverted Repeat Chimeric Gene From CMV And TuMV

Cucumber Mosaic Virus and Turnip Mosaic Virus are two major virus pathogens infecting vegetable crops in the most of the world and they often cause serious yield losses every year. Due to the lack of resistant gene present in natural germplasm, the proceeding of traditional plant breeding is slow. In recent years the rapid developments of plant genetic engineering and molecular biology have provided new ways for controlling virus infection and the possibility of protecting plants from diseases. Recent researches have revealed that invert repeat virus genes (IR) to the host plants in the transcription could engender hpRNA which is considered as an effective way to induce gene silencing degrading mRNAs homologous to the target genes.In this study, depended on the molecular clone technique, chimeric fragments with the conserve regions of CMV 2b and TuMV CP were inserted into the plant binary vector pBI 121 in the invert repeat way. And then the recombinant vector was used to infect Arabidopsis thaliana and 49 Caixin (Brassica campestris L.var. Parachinesis) respectively, and consequently transgenic plants of Arabidopsis thaliana and 49 Caixin were obtained. The results are as follows:(1) The CP genes of eight CMV isolates from different areas and vegetables in China were amplified and sequenced, respectively. The results of amino acid sequences homology showed that all the eight CMV isolates reported in this study belonged to the subgroup I of CMV which are consistent with the results of serological detection and the sequence analysis of 2b gene of CMV. However, in this study, we found that the identity of nucleotide between CP gene of CMV-BF and CMV-BG is 92.8% while that of 2b gene is 100%.Therefore we concluded that CP gene of CMV has a higher natural variation than 2b gene of CMV.(2) The conserved region sequences of 2b gene of CMV and CP gene of TuMV were cloned and formed genetic chimera inserting into the plant binary vector pBI 121 in the invert repeat way. The recombinant vector was named as pBI TC3 which was introduced into Agrobacterium tumefaciens strain AGL0 by Triparental mating.(3) The pBI TC3 vector was used to infect Arabidopsis thaliana by Floral Dip, and 56 transgenic plants of Arabidopsis were obtained. The results of PCR analysis indicated that the genetic chimera was integrated into the genome of the transgenic plants, and Floral Dip is a high-efficiency and convenient way of transformation in Arabidopsis.(4) The pBI TC3 vector was also used to infect 49 Caixin via tissue culture and 8 transgenic plants were obtained with 1.6% transformation rate.