The Prokaryotic Expression And Antibody's Preparation Of Resistance Genes To Uncinula Necator Of Chinese Wild Vitis Species

This paper presents that Stilbene Synthase (STS) is a key enzyme of the synthsis of resveratrol in plants. Since the gene was cloned , the related studies of STS gene engineering becomes a successful example of the gene engineering of secondary metabolism .The Aldehyde dehydrogenase (ALDH) is an enzyme widely existed in animals, plants and microorganisms ,it engage in many important metabolism actions ,but its function in disease resistance of plants still needs more study . In our lab , By mRNA DDRT-PCR (mRNA differential display reverse transcription-PCR ) and RACE (Rapid amplification of cDNA ends ) , the STS gene and ALDH gene of Chinese wild grape was cloned and separated from Vitis pseudoreticulata Baihe-35-1 , which has a high resistance to Uncinula necator schw.Burr.On this basis , we try to test and verify the gene's function from the way of gene's expression in E.coli , so that this could supply a basis for the STS gene's further study and use in gene engineering . Some results of this study are as follows : 1. Based on the cDNA full sequence of STS gene and ALDH gene of Vitis pseudoreticulata , we respectively designed a pair of specific primers , and obtained two DNA bands of about 1200 bp and 1700bp length respectively by PCR from the first strain of 5'RACE cDNA of Vitis pseudoreticulata Baihe-35-1 . The sequencing result shows that the factual length of the open reading frames are1179 bp and 1611bp and respectively their sequence is identical to the known STS gene and ALDH gene of Vitis pseudoreticulata , so we obtained the full sequence of STS gene and ALDH gene in vitro. 2. The ALDH gene was subcloned into the expression vector pGEX-4T-1. The recombinant expression vector was digested by BamHI and SmaI and the result shows that the ALDH gene was correctly linked to the pGEX-4T-1.The recombinant expression vector pGEX-ALDH was transformed into E.coli BL21 and induced by IPTG ,but cells had not any special expression . So we retransformed the pGEX-ALDH into E.coli BL21(coden plus) and induced by IPTG ,the cellsspecially expressed a protein of about Mr. 85 kD displayed in SDS-PAGE gel , it is identical to the GST-ALDH fusion protein we had calculated , so the ALDH gene was expressed in E.coli BL21 (coden plus). 3. The STS gene was subcloned into the expression vector pGEX-4T-1 . The recombinant expression vector was digested by BamHI and XhoI and the result shows that the STS gene was correctly linked to the pGEX-4T-1 . The recombinant expression vector pGEX-STS was transformed into E.coli BL21 cells and was induced by IPTG , the cells specifically expressed a protein of about Mr.66kD displayed in SDS-PAGE gel , it is identical to the known GST-STS fusion protein , so the STS gene was expressed in E.coli BL21 . We also fixed the best expression condition by compare the expression result of different IPTG density and cell induced culture times . 4. The GST-RS fusion protein was expressed in inclusion body , we dissolved the inclusion body by high density denaturant and renatured the protein by gradual dialysis low density denaturant . The renatured protein could be purified by Glutathione Sepharose 4B Fast flow . It supply a basis for further study of the gene's function . 5. We also used the washed inclusion body to immunolize rabbit to prepare polyclonal serum of GST-STS . Western blotting shows that the serum (diluted to 1:3000) had specific antigen-antibody recognition to the renatured GST-STS fusion protein . It supply a basis for the antiserum's more use in the localization of STS in plants and the testing of trans-STS gene plants .