Transformation Of Gscbrlk Gene And GsST2Gene Into Medicago Sativa Nonging No.1and Salt Tolerance Analysis In Transgenic Plants

With the increasing world population and declining arable land, making full use of land resources to increase food production has become a pressing task. Environmental factors such as low temperature, drought and high salinity can damage foliage seriously or even induce to death. Those factors greatly limite crop production and geographical distribution, and have become an important element constraining modern agricultural production. One of the significant means to solve this problem is to improve crop resistance to osmotic stress.Medicago sativa L. is an economically important feed crop, often described as "the king of pasture ". Besides being an important forage crop, it can be used for both fuel and phytoremediation. Alfalfa can be grown as a renewable replacement for petroleum-based products, its growth reduces the need for other non-renewable resources, such as phosphorus and nitrogen fertilizers, and it can also be used to remove contaminants from both land and water.Plant stess tolerance is a multigenic affair, and the introdiction of a single functional gene is unlikely to improve crop resistance. However, some key genes act as nodes in the stress response gene regulation network, little change in expression of which may cause more genes response by regulatory networks, where a little change in expression may cause many more downstream genes to alter their activity, thereby, achieving results that would improve crop resistance comprehensively.Wild soybean (Glycine soja) is characterized by a better stress-resistance and adaptive capacity than cultivated soybean. Over expression of GsCBRLK or GsST2in transgenic Arabidopsis resulted in enhanced plant tolerance to high salinity and ABA. In this study, two plant expression vector regulated by35S promoter are constructed to transfer the target genes into Medicago sativa Nongjing No.1separately by Agrobacterium-mediated method, named as pBEOCBRLK and pBEOMST2. Many resistant seedlings of GsCBRLK and GsST2have been obtained. The result of PCR and RT-PCR showed that the target genes were normally expressed in transgenic plants. The main results are summarized as follows:1. The acquisition of transgenic Medicago sativa L.We transformed Medicago sativa Nongjing No.1with plant expression vectors pBEOCBRLK and pBEOMST2by Agrobacterium-mediated method transformation, obtaining110pBEOCBRLK transformants, the rate of PCR detection being46%, and46pBEOMST2transformants, the rate of PCR detection being39%. Some of these plants were then analyzed through RT-PCR to determine whether GsCBRLK and GsST2were being transcribed normally. A large number of GsCBRLK and GsST2transgenic plants were then propagated vegetatively for further analysis of salinity tolerance.2. The analysis of salinity tolerance of Medicago sativa Nongjing No.lIn order to study the salt tolerance of Medicago sativa Nongjing No.1and its physiological responses under salt stress, 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 tMedicago sativa Nongjing No.1under low concentration of NaCl, it 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. The analysis of salinity tolerance of transgenic Medicago sativa Nongjing No.lAfter GsCBRLK and GsST2transgenic Medicago sativa Nongjing No.l were stressed with0,300and400mmol/L NaCl, we compared transgenic Medicago sativa to non-transfenic Medicago sativa L. by assessing specific physiological indicators. We measured the relative membrane permeability, the content of chlorophyll, the content of MDA, the activity of SOD, which were stressed with300mmol/L NaCl, and the death rate which were stressed with400mmol/L NaCl, where results showed that the transgenic Medicago sativa had a greater tolerance to salinity stress compared with non-transgenic plants.