Obtaining Of Marker-free Transgenic Potato Resistant To Late Blight

Potato is the world’s third most important food crop, after the wheat and rice. Potatolate blight caused by Phytophthora infestans is one of the most serious diseases to decreasethe potato production, which spread widely in the vast majority of potato cultivation areain the world. P. infestans is still difficult to control by genetic breeding and chemicalmethods today. Transgenic technology provides a new chance for the control of the potatocultication. In this research, R1, RB and R3a were separately transferred into potato cv.Desiree by marker-free transgenic assay, and the aim of this research is to obtaintransgenic potatoes with biological safety. The main results were displayed as follows:1) Construction of marker-free vector pMF-LIC. LIC fragment of plasmid pJG100amplified by PCR with primers OXN037/OXN038was digested with Xba I/Sac I and wasligated to the plasmid pMF-GUS digested with the same enzymes. Then the ligationproducts were transformed into E. coli DB3.1by heat shock. The positive marker-freevector pMF-LIC were verified by PCR and enzyme digestions.2) Cloning of resistance genes into marker-free vector. The resistance genes (R3a, R1and RB) to late blight were amplified from plasmid pBINPLUS-R3a, pCLD04541-R1andpCLD04541-RB with primers OXN045/OXN046, OXN051/OXN052and0XN093/OXN096respectively. The late blight resistance genes R3a, R1and RB werecloned into pMF-LIC digested with Apa I through ligation independent cloning (LIC)technique, and then were transformed into E.coli TOP10by heat shock. The recombinantvector pMF-R1/RB/R3a were verified by PCR and enzyme digestions. This methodsverfied that the resistance genes wrer successfully cloned into vector maker.3) Obtaining of the engineering Agrobacterium to late blight. The recombinantplasmid pMF-R1, pMF-RB and pMF-R3a were transformed into Agrobacterium AGL0with the help of strain HB101. The engineering Agrobacterium AGL0-pMF-R1, AGL0-pMF-RB and AGL0-pMF-R3a were constructed successfully verfied through PCR andenzyme digestion. 4) Development of the marker-free transgenic potato resistant to late blight. Thegenes R3a, R1and RB resistant to late blight were transformed into potato cv. Desireemediated by Agrobacterium tumefaciens, and4plantlets of R3a positive transgenicpotatoes were obtained and verified by PCR. The positive R1and RB transgenic potatoesare being further characterized and verfied.The transgenic potatoes will benefit theoretical research and field practice for thepotato late blight control by R gene polyculture in the field in the future.