Expression Of ABP9Enhances Tolerance To Drought Stress In Transgenic Alfalfa

Alfalfa (Medicago sativa L.) known as "Queen of Forages", is a crucial leguminous forage crop because it is rich in nutrition and hay yield is very high. Alfalfa has a long cultivated history and the most widely distribution in the word, which plays an important role in the animal husbandry. Researchers have been committed to improving the quality of alfalfa through tissue culture and genetic transformation, and cultivating new species with stress resistance, herbicide resistance, pest-resistance in recent years. Research on drought tolerance of alfalfa has been transferred from the drought resistance to transgenic drought resistance breeding as the development of molecular biology and plant genetic transformation technology.Using biotechnology to isolate the resistant genes and using transgenic method to transform these genes into alfalfa to improve the resistant of it is an effective way to cultivate new varieties of alfalfa. ABP9encodes a transcription factor of bZIP family capable of trans-activating the expression of a downstream reporter gene by specific binding to the ABRE2motif in yeast cells. ABP9plays a critical role in regulating ABA responses and modulation of the cellular levels of ROS.In this study, we translated ABP9into alfalfa by Agrobacterium-mediated infection. Bar gene was used for selectable marker, we obtained the transgenic plants which has an enhanced drought tolerance. The main results are as follows:1. Transformation of transcription factor ABP9gene into alfalfaWe used a plant expressing vector pCRI20-35S-ABP9, this vector was driven by35S promoter and has bar gene as the selectable marker. ABP9gene was transformed into alfalfa by Agrobacterium-mediated infection. Using this method, we got120regenerated plants belongs to18independent lines.2. Molecular identification of the transgenic plantsTransgenic plants were analyzed by0.5%basta spraying, most lines showed normal growth and green leaves, but the leaves of wild-type plants were turned to yellow and even dead. These18independent lines were identified though PCR. RT-PCR in molecular levels. The results indicated that16lines can be ampified ABP9specific fragment, and this gene got effective expression in alfalfa, and expression levels are different in different lines.3. Drought tolerance identification of the transgenic plantsTransgenic plants were not watered to identify its drought tolerance. Under drought stress, transgenic plants showed great drought tolerance. The transgenic plants were still able to maintain green, only some leaves began to wither after20d treated with drought stress, but at this time the wild-type plants showed wilt.4. Physiological and biochemical determination of transgenic plants under drought stressTest results showed that the related water content, chlorophyll content and proline content of transgenic plants are higher than the wild-type plants. While the malondialdehyde (MDA) content and hydrogen peroxide content (H2O2) were significantly lower than wild-type plants. All these results indicating that over-expression ABP9gene in alfalfa can more effectively discharge the toxic of ROS. which can improve the drought tolerance of plants.