Cloning And Functional Analysis Of Dehydroascorbate Reductase DHAR Gene In Wheat

Vitamin C,also called as ascorbic acid（AsA）,can remove toxic reactive oxygen species（ROS）in cells,effect cell elongation and division and participate in xanthophyll cycle and regeneration of vitamin E.It has important antioxidant and metabolic functions in animals and plants.For humans,vitamin C can only obtained regularly form daily diet,because it can neither be synthesized nor stored in the human body.Therefore,increasing the content of vitamin C in plants is of great significance to human health.Dehydroascorbate reductase（DHAR）is the key enzyme in the ascorbate metabolism cycle.It can catalyze the reduction of glutathione and reduces the ascorbate metabolism intermediate dehydroascorbic acid（DHA）to ASA,thereby reducing the loss of AsA and increasing its content.In this experiment,wheat was used as the experimental material,the DHAR gene of wheat was cloned,and the Ubi-DHAR overexpression vector was constructed.The positive plants were obtained by gene gun transformation,tissue culture,PCR positive verification,etc.The transgenic plants of T1generation were tested at the level of gene expression,ascorbic acid content and enzyme activity.At the same time,drought stress and oxidative stress were tested on the plants,which was the stress resistance was tested and studied.In this study,the DHAR gene in wheat was overexpressed to improve its vitamin C content and increase its nutritional value.In addition,it enhanced the resistance of wheat and provided ideas for plant resistance breeding.The main results obtained are as follows:1.Three copies of the DHAR gene were cloned from wheat,and a copy with a higher expression level was selected to construct an overexpression vector（Ubi-DHAR）with Ubi as a promoter.2.The vector（Ubi-DHAR）was transferred into immature embryos of wheat by gene gun transformation technology.After transgenic tissue culture and positive identification by PCR,the transgenic wheat plants were obtained.3.Molecular and physiological experiments analyzed DHAR gene expression,DHAR enzyme activity,AsA content and AsA/DHA in overexpressed transgenic plants.The results showed that DHAR gene expression in leaves and seeds increased in different degrees,while the content of AsA and AsA/DHA increased more significantly in leaves compared with seeds.The DHAR enzyme activity of the transgenic plants was increased about 2.5 times as much as that of wild type plants.4.The preliminary stress resistance test of T₁ transgenic plants showed that the drought resistance and antioxidant capacity of the transgenic plants were improved to some extent than the wild type.Preliminary results indicate that overexpression of DHAR gene and increased AsA content are important for improving the resistance of wheat.