Expression Of OsAPX2 Enhances Tolerance To Salt Stress In Transgenic Alfalfa Plants

Alfalfa (Medicago sativa L.) known as "the king of grass", 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. In recent years, global food security is getting worse and worse, especially in China. More and more fertile land were used for food production, so forages like alfalfa has to find new cultivation. If salinity, decertify, drought and water shortage areas can be exploited for alfalfa planting, there is a chance to find a balance between forage production and our country’s food security, which will bring enormous benefits to our country’s food security and livestock development.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. In this study, we cloned the OsAPX2 gene from rice, and translated it into alfalfa by Agrobacterium-mediated infection. Bar gene was used for selectable marker, we obtained the transgenic plants which has an enhanced salt tolerance. The main results are as follows:1. Transformation of ascorbate peroxidase (OsAPX2) gene into alfalfaOsAPX2 gene cloned from rice was conducted a plant expressing vector OsAPX2-pCambia3301, this vector was driven by 35S promoter and has bar gene as the selectable marker. OsAPX2 gene was transformed into alfalfa by Agrobacterium-mediated infection. Using this method, we got 100 regenerated plants belongs to 22 independent lines.2. Molecular identification of the transgenic plantsTransgenic plants were analyzed by 0.5% basta spraying, most lines showed normal growth and green leaves, but the leaves of wild-type plants were turned to yellow and even dead. These 22 independent lines were identified though PCR, RT-PCR and Southern blot in molecular levels. The results indicated that 20 lines can be amplified OsAPX2 specific fragment, and this gene got effective expression in alfalfa, and the copy number and expression levels are different in different lines. 3. Salt tolerance identification of the transgenic plantsTransgenic plants were exposed to 200 mM NaCl to identify its salt tolerance. Under salt stress, transgenic plants showed great salt tolerance. The transgenic plants were still able to maintain green, only some leaves began to wither after 25d treated with salt, but at this time the wild-type plants showed wilt. Water desalination experiments showed that transgenic plants can restore and grown well but the wild-type plants can not.4. Physiological and biochemical determination of transgenic plants under salt stressTest results showed that the related water content, chlorophyll content and proline content of transgenic plants are higher than the wild-type plants, especially the proline content increasing significantly. While the electrolyte leakage, malondialdehyde (MDA) content and hydrogen peroxide content (H2O2) were significantly lower than wild-type plants. All these results indicating that over-expression ascorbate peroxidase (APX) in alfalfa can more effectively discharge the toxic of ROS, which can improve the salt tolerance of plants.