Transformation Of GsbZIP33and GsCBRLK Gene Into Medicago Sativa L. Cv. Zhaodong And Salt Tolerance Analyais In Transgenic Plants

Salinity stress is one of the major environmental factors that limit plant growth and development, adversely affecting crop production. With the rapid development of molecular biology and bioengineering, using the bioengineering strategies to improve stress tolerance in crops has been an important aspect of modern agricultural research. However, stress tolerance is a complex physiological process in which many genes are involved. Using the bioengineering strategies to transfer a key controlling gene (eg:kinase gene, responding and transducting the stress signal) is a more efficient way to slove the problem.Medicago sativa L. is an important leguminous forage crop worldwide, and it not only supplies abundance of forage for animals but also improves soil fertility. However, salinity problems in agriculture represent a major constraint in the productivity of crops and forage pastures. Therefore, breeding salt tolerant Medicago sativa cultivars is very necessary for this important forage crop adapting to saline soils. Salt tolerance of Medicago sativa can be improved by conventional breeding methods, but it takes a long time to select salt tolerant plants. With the rapid development of molecular biology and bioengineering, using the bioengineering strategies to improve stress tolerance in Medicago sativa has been an important aspect.Wild soybean (Glycine soja) is characteristic of better stress-resistance and adaptive capacity. It is an important gene resource in molecular breeding by means of transgenic technology. In this study,we use four Medicago sativa L.cv.Zhaodong as material, construct of plant expressive vectors of GsbZIP gene and GsCBRLK gene that regulated respectively by El2and CaMV35s promoters by Agrobacterium Tumefaciens. Many transgenic plants were obtained. The research work will provide the function for cultivating transgenic variety that resist to salt stress and studying salt stress resist mechanism. The main results were summarized as follows:1. Identification of transgenic Medicago sativa plantsWe transformed Medicago sativa Zhaodong with GsbZIP33and GsCBRLK gene by Agrobacterium-mediated method transformation. Including101resistant plants by transfomating GsbZIP33, there are55PCR positive plants, so positive rate is54%; Including75resistant plants by transfomating GsCBRLK, there are36PCR positive plants, so positive rate is48%. The results of PCR and RT-PCR showed that the purpose gene was integrated into Medicago sativa genome and overexpressed in transgenic plants.2. Establishment of salt tolerance identification system for Medicago sativa ZhaodongIn order to study the salt tolerance of Medicago sativa L.cv.Zhaodong, seedlings were cultured in1/2Hogland solution supplied with NaCl at different concentrations of0,50,100,200,300and400mmol/L, respectively. Salt injury was measured after the treatment. The content of malondialdehyde (MDA), chlorophyll, proline before and after3,6,9,12,15and21d of salt stress were measured and analyzed, respectively. The results showed that there was no significant effect on the growth of two Medicago sativa under low concentration of NaCl, they had certain salt tolerance ability. The content of MDA increased with the increase of NaCl concentration. While under the stress of200and300mmol/L, the content of MDA increased primary stage then decreased, and then increased with time prolonging. With the concentration and duration of NaCl stress increasing, salt injury was increased, the content of chlorophyll reduced, and proline content increased remarkably. But the accumulation of proline was not entirely consistent with their salt-tolerant performance.3. Over-expression of GsbZIP33, GsCBRLK increases salt tolerance of alfalfaTransgenic Medicago sativa plants overexpressing the GsbZIP33, GsCBRLK showed enhanced salt tolerance. Transgenic Medicago sativa grew well in the presence of300mM NaCl for15days, while wild-type plants exhibited severe chlorosis and growth retardation. Although transgenic Medicago sativa grew slowly and even appeared yellow leaves in400mM NaCl treatment, wild-type plants exhibited chlorosis and even most of wild-type plants died. In addition, Samples from transgenic and wild-type plants treated with300mM NaCl for0,3,6,9,12and15days were selected for physiological analysis. Lower membrane leakage and MDA content were observed in transgenic alfalfa by contrast with wild-type plants during salt treatment. Moreover, reduction of chlorophyll content in transgenic alfalfa was less than that in wild-type plants. Furthermore, the transgenic plants showed enhanced activities of SOD, when compared to wild-type plants. These results indicated that the expression of GsCBRLK confers enhanced tolerance to salt stress in transgenic alfalfa.