Constructing Transgenic Tomato For High Resistance Against TYLCV By RNAi Mediated Multi-gene Silence

Tomato (Lycopersicon esculentum Mill.), as one of the most important vegetable crops in the world, has more than 850,000 hectare of planting area in China and obvious economic value. However, recently, the virus disease, such as tomato yellow leaf curl virus (TYLCV), has spread quckily and lead to severe damage in China. Culturing resistant variety was one of the efficient methods to prevent and control the disease. In this study, by cloning the objective gene, combing with RNAi, and constructing the chimeric gene vector to transform to the plants sensitive to TYLCV, we selected the new tomato variety highly resistant to TYLCV by indentifying the resistant viability. The results were as following:The AV1, AC1 and AC3 gene were cloned as objective gene. By homology analysis, the positive and reverse conserved sequences,151bp of AV1 (893Bbp-1044bp),150bp of AC1 (1710bp-1860bp) and 153bp of AC3 (1054bp-1207bp), were linked to interference vector pBIN19 to construct three RNAi vector, pBIN19-AV1 (i/r), pBIN19-AC1 (i/r) and pBIN19-AC3 (i/r). By overlap extention PCR, the three gene fragments were assembled to the chimeric gene AV1-AC1-AC3. Then the positive and reverse sequences of the chimeric gene were linked to the interference vector pBIN19 to construct the RNAi vector pBIN19-AV1-AC1-AC3 (i/r).The RNAi vector of tomato PDS gene, pBIN19-PDS (i/r), was constructed and transformed to EHA105 to infect the 2-3 leaf tomato seedlings. The results showed that the infected tomato leaves showed whitening symptoms, and distributed systemly on the new true leaves, which suggested that the transient expression system was viable in tomato. Infection of tomato with pBIN19-AV1 (i/r), pBIN19-AC1 (i/r), pBIN19-AC3 (i/r) and pBIN19-AVl-ACl-AC3 (i/r) showed that the four RNAi vectors could have resistant effect, in which the resistant viability of the chimeric gene AV1-AC1-AC3 was the highest. The resistant rate could reached 100% in 5 weeks.The regeneration and transformation systems were established and optimized. The results showed that the cotyledon and hypocotyls could be the explants of tomato genetic transformation. The induction rate of callus and adventitious buds of the two explants were similar. The most suibable medium of positive buds of cotyledon was MS+0.5 mg/L IAA+0.5 mg/L BAP, while that of hypocotyls was MS+0.5 mg/L IAA+2.0 mg/L BAP. The most proper medium for rooting was MS+0.1 mg/L IAA. The induction of adventitious buds reached the peak (39%) after infection with agrobacterium (OD600=1.0) for lOmin and cultured 2d under dark condition, while the induction of hypocotyls reached the peak (40%) after infection with agrobacterium (OD600=1.0) for 30min and cultured 2d under dark condition.By analyzing the resistance to virus of TO plants, we found that the resistant ability of transgenic plants of chimeric gene was better than that of single gene.