GroEL Gene Expressed In The Different Promoters And DsRNA Mediated Virus Resistance To TYLCV In Tobacco Plants

Geminiviruses are a group of single-stranded circular DNA viruses occurred worldwide,and caused severe diseases in several economically important crops.In China, Several kinds of begomoviruses were identified in tomato,tobacco and squash in Yunnan and Guangxi province.Here, GroEL gene was constructed three expression vectors which isolated from endosymbionts of Bemisia tabaci,and AC1,AC2 repeat gene was constructed RNA interference expression vector which comes from Tomato yellow leaf curl virus(TYLCV).The vectors were transformed into Agrobacterium tumefaciens,resistance of these transgenic tobacco plants were evaluated.Get the results as below:1. With the purpose of enhancing the resistant plants against TYLCV, the GroEL gene which driven by CaMV35S, SUC2, PGHNBS promoters was exploited from Bemisia tabaci. Three plant expression vectors (P-GroEL,P-SUC2-GroEL,P-PGHNBS-GroEL) were successfully constructed.2. P-GroEL,P-PGHNBS-GroEL,P-SUC2-GroEL were transformed into Agrobacterium tumefaciens LBA4404.The positive clones confirmed by PCR and digestion were used for transformation of tobacco plants.The vectors were transformed into tobacco by Agrobacterium tumefaciens -mediated method via leaf disc.Some kanamycin-resistant transgenic plants expressing GroEL gene were obtained. The result of RT-PCR proved that GroEL gene was expressed in three regenerated Kanamycin resistant tobacco plants. Virus infection at the first time, 19 resistant tobacco plants acquired: 7 plants were transformed with expression vector P-GroEL, P-PGHNBS-GroEL and P-SUC2-GroEL separately have 6 resisitant tobacco plants. Following, virus infection at the second time, it was shown that 7 resistant tobacco plants acquired: only one of them transformed with expression vector P-GroEL. However, 3 resisitant tobacco plants transformed with expression vector P-PGHNBS-GroEL and P-SUC2-GroEL, separately. Via twice inoculated experiment, comparing with CaMV35 promoter, it was obvious that transgenic tobacco with PGHNBS and SUC2 promoters expressed more resistance toward TYLCV.3.The AC1 and the AC2 repeat segment of the TYLCV was cloned and the sense segment,intron and the antisense segment were constructed into plant expression vector pBI121 by using a series of cloning methods.The vector has a reverse repeated sequence as a foreign segment.The segment should match between the reverse sequence and torm a short hair-pin RNA(shRNA) after the transformation of vector. 4.The expression vector pBI121-AC was transformed into tobacco by Agrobacterium tumefaciens LBA4404.Callus was screened through Kanamycin and shoot growth into transgenic tobacco.PCR confirmed that AC1,AC2 repeat gene had been inserted into tobacco genome DNA.