Transformation Of Yeast Rad52 Gene Into Arabidopsis And Techniques For Transformant Screening

Gene targeting has become an indispensable reverse genetics tool for studying gene function, but in plants transforming DNA integrates mainly in a random manner into the genome. Tremendous efforts have been made to improve the efficiency of gene targeting, but it has not become a practical technique. Transforming DNA almost exclusively integrates into genome via homologous recombination in yeast, which results in high efficient gene targeting. It is supposed to be an important approach to improve plant gene targeting efficiency by introducing the homologous recombination machinery of yeast into plants. Rad52p is a crucial enzyme to homologous recombination in yeast, and most plants lack it. In order to study the effects of Rad52 gene on homologous recombination and gene targeting in plants, Rad52 gene was isolated from yeast by PCR and transformed into Arabidopsis.Because gene targeting efficiency is very low in plants at the moment, it is necessary to establish an efficient transformation system to study gene targeting. Agrobacterium mediated in planta transformation of Arabidopsis was well established, and is tissue culture free for transformation. But the transformant screening process still requires tissue culture, which is labor-intensive, and material lose often occur because of contamination. Here we established three simple but efficient screen methods free from aseptic culture.The main resultes are summarized as follows:1. Rad52 gene were isolated from yeast and Agrobacterium binary vector were constructed. Rad52 gene were transformed into Columbia ecotype of Arabidopsis by Agrobacterium-mediated floral dip method. Transgenic plants were obtained and screened by PCR. The results indicated that the transformed gene was integrated in the Arabidopsis genome.2 Three different methods for screening Arabidopsis transformants free from sterilized culture have been developed. The first one is to screen seeds on filter paper moistened by liquid 1/4MS macronutrients with selection agent and 50μg/ml ampicilin. The second method is to screen on water agar, which is based on the traditional tissue culture method, but the medium only contain 1/4 MS macronutrients with selection agent and proper antibiotics solidified by 0.8% agar. The low nutrient and antibiotics make sterilization unnecessary. In the third method, seeds are germinated for 1.5-2 day in liquid 1/4MS macronutrients with selection agent before being transferred to the soil. Seedlings survived screening grow the best and are easier to be transplanted. The three methods are all simple and reliable, which resolves a bottleneck problem in Arabidopsis in planta transformation.