Study On The Transforming The Plant-derived Gene Resistant To PVY To Tobacco

Potato virus Y (PVY) was reported firstly by Smith in potato in 1931.It is widespread all over the world now. PVY can infect many crops, such as tobacco, potato and capsicum. Tobacco disease caused by PVY is known as yellow spot necrosis, vein strip disease or vein necrosis. It is not uncommon in the tobacco planting areas of the world containing our country. In recent years, the frequency and destruction of PVY on tobacco have been increasing rapidly in our country. It has been becoming important on tobacco production to solve and control this virus disease. When they are cosmically planted, the transgenic lines via the ectopic expression of different virus genes derived from the same virus would lose their resistance because they can cross each other and their offspring contain two or more than two kinds of virus genes. Moreover, the virus gene can be translated into the protein which would be harmful to human. In this study, we transformed the plant-derived gene to tobacco. It can improve the resistance, and also solve the biologic risk brought by translating the virus gene into the plant tissue. So it has the far-reaching potential development.The pvr2 is deemed to the resistant gene to PVY in capsicum. Recent studies have proved that this gene is a allele of the eif4E. The protein expressed by eif4E-pvr2 gene can combine with the vpg protein of PVY. It can affect the PVY self-replication, which reduces the accumulation of PVY particle and impairs the symptom of the disease. Ruffel et al.(2002) had cloned the eif4E-pvr2 gene from the wide type capsicum variety, Yolo Y. They transformed this gene to the variety Yolo wonder which is susceptive to PVY, and this transgenic variety achieved the resistance to PVY.In this study, according to the CDS sequence of eif4E reported in the GenBank, we designed the PCR amplification primers, and cloned 689 bp susceptive eif4E gene fragment from capsicum leaves via RT-PCR. eif4E gene was compared with eif4E-pvr2 gene CDS sequence reported in the GenBank. The result indicated that the most segment are homological, but they have two different bases at 200 bp and 236 bp, which is A and G for eif4E-pvr2 and T and T for eif4E respectively. Based on this difference, we cloned eif4E-pvr2 gene via point mutation. We constructed the plant expression vector pEIF2301 and pMEIF2301, according to eif4E gene and eif4E-pvr2 gene respectively. We transformed eif4E and eif4E-pvr2 to tobacco variety K326. and, achieved 10 lines and 12 lines with kanamycin resistance respectively. We have 4 positive lines via GUS detection and PCR, which indicates that eif4E and eif4E-pvr2 are integrated into the tobacco genomic gene.