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Abstract To reveal the disease-resistant mechanism of Chinese wild grape transcription factor in the process of grape resistant to powdery mildew, the highly resistant line Baihe-35-1 of Chinese wild Vitis pseudoreticulata W.T. Wang was used as the materials, and a 1 053 bp full-length cDNA encoding V. pseudoreticulata RING-finger protein1 gene (VpRFP1) was obtained using the transcript product induced by grape powdery mildew (Unicunula necator (Schw.) Burr.) at seven different stages as templates. The resultant PCR products were cloned to pMD19-T vector, sequenced and subcloned into pGEX-4T-1 vector. The recombinant expression clone were transformed into Escherichia.coli BL21 and induced expression by IPTG. A 64 kD fusion protein GST-VpRFP1 was obtained with the form of inclusion body. The fusion protein was purified by electrodialysis, and immunized the rabbit to obtain the polyclonal antibodies. The immunity-antigen reaction was tested by Western blot, showing a specific antigen-antibody recognition feature. The results suggested that the fusion protein GST-VpRFP1 was expressed at high levels 4 h after 0.1 mmol/L of IPTG induction (27℃). And the polyclonal antibodies obtained by immunized rabbit with the purified fusion protein can be used for the functional analysis of VpRFP1 gene.
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Received: 29 April 2010
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