Function Identification Of Gene VaERD15 From Chinese Wild Vitis Amurensis

Grapes have a very wide range of cultivation both in China and in the world. Most cultivars in production belong to Vitis vinifera, which has good fruit quality but poor cold-resistance and don’t suit to plant in cold region of northern China. The Chinese wild Vitis amurensis is known as the most cold-resistant species, which can survive at-40℃. If the new cultivar with good traits of Vitis vinifera and cold resistance of Chinese wild Vitis amurensis is breeded and applied, the scope of planting grapes can be expanded. Therefore, the study of cold-resistant genes associated with Chinese wild Vitis amurensis and transgenitic breeding shows a great importance.VaEDR15, a cold-resistant related gene, which cloned from Chinese wild Vitis amurensis,was transferred to Arabidopsis thaliana and Vitis vinifera cv. ’Red Globe’, respectively. After the low-temperature treatment, the transgenetic plants either Arabidopsis thaliana or ’Red Globe’ grape were tested in molecular level and in phenotype, as well as in physiological and biochemical indexes. The function of VaEDR15 has been initially validated through comparison with non-transgenetic plants. The results obtained are as follows:1. Assay of cold-resistance for transgenetic Arabidopsis thaliana plants with VaEDR15 gene(1) VaEDR15 has been transferred to Arabidopsis thaliana through Agrobacterium tumefaciens mediated method. The transgenetic plants have been picked out through PCR and Southern blot analysis. Two of transgenetic plants were selected and treated under low-temperature(-6℃), and the leaves were taken and as samples after cold treatment with 2,4, 8, 12, 24, 48, and 72 hours. The results showed that the expression of VaEDR15 increased with the stress time by fluorescent quantitative PRC and western blot analysis, but the non-transgenetic Arabidopsis thaliana plants remained the same as before.(2) Freezing injury occured both in wild type and in transgenetic Arabidopsis thaliana plants after low-temperature stress, but the extent of the injury and the numbers of seedling damaged were different. The former was more severe than the latter.(3) Some physiological and biochemical indicators related to cold-resistance including relative conductivity, soluble sugar, soluble protein, proline, malondialdehyde and enzymeactivities of superoxide dismutase, superoxide and catalase were measured in transgenic Arabidopsis plants. The results showed that the transgenic plants are more resistant to cold than the wild-type plants.2. Assay of cold-resistance for transgenetic ’Red Globe’ grape with VaEDR15 gene(1) VaEDR15 has been transferred to proembryogenic mass(PEM) of ’Red Globe’ grape through Agrobacterium tumefaciens mediated method. 10 positive plants have been screened out through medium filled with basta, PRC and Southern blot.(2) The tube seedlings of transgenetic plants were propagated. The two similar well-grown transgenetic plants had been selected and dealt with low-temperature. Then, the expression analysis of VaERD15 in transgenetic plants was performed by fluorescent quantitative PRC and western blot analysis. The results showed that there was an obvious increase of VaEDR15 expression in transgenic plants with the stress time, but the change of VaERD15 expression in wild-type plants was not obvious.(3) The physiological and biochemical indicators related to cold-resistance including relative conductivity, soluble sugar, soluble protein, proline, malondialdehyde and enzyme activities of superoxide dismutase, superoxide and catalase were measured in transgenic ’Red Globe’grape. The results showed that the transgenic plantsis more resistant to cold than wild-type plants.The above results indicated that compared with the wild type, the transgenic Arabidopsis and transgenic ’Red Globe’ plants presented stronger cold resistance. This means that the VaERD15 gene may play an important role in the process of cold resistance of Vitis amurensis.