The Gene Cloning And Genetic Transformation Of Sugarcane Mosaic Virus Coat Protein Gene

Sugarcane mosaic virus (SCMV) is a very serious virus disease for sugarcane growth that can cause serious yield lost. Farming practice and chemical control is not sufficient enough to control this disease. Most research prove that traditional plant breeding has played an important role in resistant to virus by providing varieties with disease resistance, higher yield and better quality. However, the sugarcane characters and the difficulties during the selection procedure limited the progress in virus resistance breeding. Plant genetic engineering provides a new way for developing virus resistant cultivars.Primers were designed according to the amino acid conservative sequence of SCMV sub-group. The 1.8 kb desired bands were amplified by RT-PCR from the highly infected Badila by SCMV. PCR products wee purified and cloned into pMD 18-T Vector for sequence analysis. The sequencing result of this fragment shows that the sequence comprised 1581bp ORF and 231bp UTR. The ORF encoded a putative protein of full coat protein and partly nuclear inclusion b. The coat protein sequence was most similar to SCMV-D with identities of 96.08% and 97.44% at the nucleotide and acid levels respectively. Secondly it was similar to SCMV-A with homology of 94.71% and 96.81% at the nucleotide and acid levels respectively.In this research, the calluses of Badila were transformed with pNUSCP containing SCMV-CP by bombardment. After selection on the medium containing 30 mg/L G418, 53 regenerated plants were obtained. PCR analysis showed that 24 plants contained the CP gene and NPTII gene. In which 13 plants contained the CP gene confirmed by southern blotting, and could stably inherited to sub-cloning.The 13 sub-cloning of transgenic sugarcane were inoculated with SCMV for virus resistant evaluation in pest-tight-house. The results showed that the transgenic plants represent highly resistant to virus except B4 and B31 clones, and the untransgenic plants were almost infected by SCMV. By analyzing for chlorophyll. photosynthesis and enzymes in the metabolize of reactive oxygen species in transgenic and untransgenic plants, the results also showed that the transgenic plants can protect sugarcane cell from SCMV damage, but in the untransgenic plants, the tissue were seriously harmed by the accumulation of harmful reactive oxygen species because of the infection of SCMV virus.