GsSAMS Gene Transformation Into Alfalfa And Cultivation Of Transformation New Lines

Drought, salinity and low temperature are the most severe natural disasters of many abiological adversities.They can damage to plants, even induce foliage to death. They also might reduce crop yield and restrict crop planting area. The problem of lost crop yield and risk of the agro-environment lead by those factors is worldwide. Over 800 million hectares of land throughout the world are salt-affected, this is over 6% of the world's total land area. Because of excessive land clearing and irrigation, increased salinization of arable land is expected to have devastating global eVects, resulting in 30% land loss within next 25 years and up to 50% by the middle of 21st century.Alfalfa is an important feedingstuff , as " the king of pasture ", it has been as the main source of animal protein and amino acids. Alfalfa is more salt-tolerance in the legumina, and it is not only a major pasture, but to improve the soil conditions and protect the environment.In this study, through screening of Glycine soja's full-length cDNA library,one osmotic stress correlative genes were obtained through gene cloning technique. Alfalfa as a receptor material, we construct plant expression vector, which have GsSAMS gene and regulated by different promoters. And Alfalfa was transformed by Agrobacterium Tumefaciens. Many transgenic plants were obtained. This study has important research value and broad prospects for development and application.It established alfalfa molecular breeding technology system, and make full use of saline-alkali soil resources in our country. The main results were summarized as follows:1. Screening of osmotic stress correlative genes from the Glycine sojas'ESTs databaseAnalysis of Glycine soja's ESTs database under salty stress, 1 ESTs were obtained which correlative osmotic stress. It was S-adenosylmethionine synthetase (SAMS). During biochemical process, the GsSAMS genes play important roles in methyl and mercapto transduction and polyamic synthetical.2. Gene cloningNortheast wild soybean as the template cDNA, clone a total length of osmotic stress-related gene GsSAMS.3. Construction of plant expression vectors (1) Construction of plant expression vector pCS29A,which include GsSAMS gene regulated by rd29A promoter.(2) Construction of plant expression vector pCSE12,which include GsSAMS gene regulated by E12 promoter.These two plant expression vectors were used for transformation of alfalfa and molecular detection.4. Genetic transformation mediated by Agrobacterium TumefaciensThe GsSAMS gene which were regulated by promoter rd29A and E12 promoter respectively, were transferred into alfalfa by Agrobacterium mediated method, obtained many resistant seedlings.(1) The resistance rate of pCS29A resistant buds was 34.25%.(2) The resistance rate of pCSE12 resistant buds was 21.25%.5. Detection of resistance plant by molecular biology technology(1) pCS29A have been transformed into 24 resistant plants, in which PCR-positive plants is 9, PCR positive rate was 37.5%(2) pCSE12 have been transformed into 16 resistant plants, in which PCR-positive plants is 4, PCR positive rate was25%...