Cloning And Expression Analysis Of VpUSP Gen In Chinese Wild Vitis Pseudoreticulata

Universal stress protein(USP)is a small cytoplasmic bacterial protein that is upregulated when the cell is exposed to stress agents.USP enhances when the rate of cell survival during prolonged exposure to such conditions, and it may provide a general"stress endurance"activity.Grape powdery mildew is a fungal disease which seriously harms the world's grape production. In the famous grape growing areas, such as California, France, Italy, Australia and Xinjiang in China, the grape powdery mildew has down great harm and caused huge economic losses for grape production. Combination of genetic engineering and traditional breeding technology, identification of resistant gene in resistant grape germplasm and cultivating of new resistant variety are effective ways to solve the problem.Here,we described the isolation and nucleotide sequence analysis of the EST from the cDNA library constructed of powdery mildew-infected leaves of'Baihe-35-1'and compared its amino acids USP from other species. The major results of this dissertation are as follows:1.A USP(Universal stress protein) related EST sequence from the cDNA library constructed of powdery mildew-infected leaves of'Baihe-35-1'is screened. Full length of this gene is obtained by RACE and was designated as VpUSP(GenBank accession number GU946975) . It is 836bp ,which encondes 175 amino acids.We blast the sequence of Arabidopsis thaliana, Brassica rapa from GenBank,result shwoede that they shared 62%- 59%.2.Semi-quantitative RT-PCR was employed to study the expression of the gene. The results showed constitutive expression of the gene was observed in susceptible lines. However, the gene could be induced by grapevine powdery mildew in resistant lines, and reached the climax after 48h, It suggests that VpUSP has the expression activity during the powdery mildew infecting leaves.3. Different combination of plant growth regulators were studied to the"BaiHe-35-1"plantlets subculture growth condition.Result shows that the most suitable medium for the"BaiHe-35-1"plantlets subculture was1/2 MS+IBA1.0 mg/L+NAA0.1 mg/L+source28 g/L+agar0.6%.4. Leaf,petiole and stem of"BaiHe-35-1"were used in this study. Different combination of plant growth regulators were studied to induced the adventitious buds. Result shows that the best medium in inducing the adventitionous buds was MS+IBA0.2 mg/L+TDZ4 mg/L+source 15g/L+agar0.6%.And the regeneration efficiency was 9%,10%,8%。.