Establishment Of Genetic Transformation With High Frequentcy In Medicago Truncatula And Overexpression Of Gene Mt2g021690 In Arabidopsis

Medicago truncatula is an annual plant of legume, which is the third large-scale sequencing plant following Arabidopsis thaliana and Oryza sativa. As a model legume plant, M. truncatula can satisfy our maximized need of research on legume biology and gene improvement. Genetic transformation is an essential technology used in clarifying mechanism of gene expression and regulation of plant growth and development. Therefore, it is a powerful tool for studying gene function of plants. One of the purposes for the present study is to construct high-efficient regeneration and genetic transformation system in M.truncatula.microRNAs are a novel class of short, endogenous non-coding small RNAs that have base pair with their target genes to repress their translation or induce their degradation and then regulate the expression of genes. miRNAs play crucial roles in biological processes, including development, metabolisms, organogenesis and stress responses. Protease inhibitors (PI) are widely present in plants, animals and microorganism which can bind with the protease active site or allosteric site, and consequently inhibit the catalytic activity of enzyme or prevent zymogens from transforming into active enzyme. PI plays an important role in regulation of enzyme activity, signaling transduction and resistance against pathogens and insects. miR1509 is a specific small RNA in legume which target gene is classified to the protease inhibitor family.My work is mainly focused on the following three parts:(1) Using the leaves of M. truncatula as explants, different kinds and concentrations of hormones which affected the regeneration frequency were studied. An efficient system for production of transgenic M. truncatula plants were established by optimizing the various conditions including concentrations of infection, infection time, hygromycin (Hyg) concentrations, antibiotic concentrations, and co-culture time. (2) Two over-expressing vectors, miR1509 and its target gene Mt2g021690 have been successfully constructed and transferred separately into M. truncatula through Agrobacterium tumefaciena mediated approach. Thrity-eight pieces of callus with resistance were obtained, and the transformatic efficiency can be as high as 67.3% calculated by GUS staining and PCR identification. (3) An over-expression vector containing gene Mt2g021690 was transferred heterogeneticly into Arabidopsis, followed with real-time PCR analysis of the expression of this gene in T2 transformant progeny, indicating that the gene may be relative with signal pathway mediated by jasmonic acid.