Transgenic Resistance Mediated By Potato Virus Y HC-Pro Gene And Its Mutants

Potyvirus-encoded helper component-proteinase (HC-Pro) plays importantroles in viral life cycle. It is involved in polyprotein processing, cell-to-cellmovement, transmission by aphid vector. It also enhances viral gene expressionand suppresses host defense mechanism (RNA silencing). In present work, weconstructed four plant expression vectors expressing full length PVY HC-Pro geneand its three mutants. Tobacco plants (cultivar NC89) were transformed withAgrobacterium tumefaciens carrying these four vectors. Meanwhile, HC-Pro gene andone mutation, HC-C, were expressed in bacteria E. coli BL21, and antisera againstthe expressed protein were raised in rabbit. The main results of this study were asfollows:1.Three fragments, HC-C, HC-C' and HC-NC that carrying translatable and untranslatable C-terminus, translatable N and C-termini respectively, and full- length HC-Pro gene were amplified by PCR. They were digested with BamHI and KpnI, ligated into pUC18 and verified by sequencing. The three mutants and full-length HC-Pro gene were cut by BamHI and KpnI from pUC18 and then inserted between the 35S promoter and the nos terminator sequences of the binary vector pROKII to generate pROK-C, pROK-C', pROK-NC and pROK-HC. The vectors were transformed into Agrobacterium tumefaciens LBA4404 for plant transformation.2.The genes were introduced into tobacco (NC89) via Agrobacterium tumefaciens- mediated transformation. The transformed shoots were selected in MS medium contain 100 mg/L kanamycin sulfate. The regenerated plants initially were screened by PCR. The results shows that we obtained 121,116,124 and 130 transgenic plants that carrying HC-C, HC-C', HC-NC and full length HC respectively.3.The regenerated plants were inoculated using PVYN . The plants with no symptom were reinoculated two weeks later. The results indicated that 6, 15, 8 and 3 individuals of transgenic C, C', NC, and HC plants were resistant to PVYN, respectively.4.Green peach aphid (Myzus persicae) were fasted for 2 h, then allowed a 5 min acquisition access feeding on transgenic plants infected by PVYN; thereafter, they were transferred to nontransgenic tobacco seedling to transmit virus. The results indicated that plant expressing HC-N or HC-NC could reduce the efficiency of aphid transmission significantly, while transgenic plants expressing HC-C could not reduce the efficiency of virus transmission by aphids.5.The HC-C and full-length HC-Pro were cut from vector pUC18 with BamHI and SacI, and ligated into expression vector pET-22(b+) to generate pET-C and pET-HC. pET-C and pET-HC were transferred into E.coli BL21. SDS- PAGE showed that both pET-C and pET-HC could be expressed as a fusion protein to high levels when induced with IPTG. Using these fusion proteins as...