Transformation Of GsZFP1and GsWRKY20Genes Into Medicago Sativa L. And Salt And Drought Tolerance Analysis In Transgenic Plants

Abiotic stresses, especially drought, low temperature, saline and alkaline stress are majorenvironmental factors that adversely affect crop production, crop quality and geographicaldistribution in the world. Alfalfa (Medicago sativa L.) is one of the most important leguminousforage crops worldwide. It is not only large cultivation area, but also wide distribution. Alfalfahave rich protein, fiber and other nutrients such as calcium, phosphorus. It is recognizedworldwide forage crop. Although alfalfa has tolerance for cold, drought and salinity, it is onlysuitable for mild saline and drought soil. Drought and high salt have become important reason forlimiting the expansion of the alfalfa area.In previous our study, GsZFP1and GsWRKY20were cloned from Wild soybean (Glycine soja) andwere overexpressed Arabidopsis which enhanced tolerance drought. In this study, we wouldtransformed into legume alfalfa, in order to get drought tolerance transgenic alfalfa and study thefunction of these two genes in alfalfa. Specific studied include: Construction of plant expressionvector GsZFP1and GsWRKY20and genetic transformation mediated&plant regeneration;Molecular biology identification of GsZFP1and GsWRKY20resistant plants by PCR, RT-PCR andqPCR; Overexpression of GsZFP1and GsWRKY20were analyzed phenotypic and physiologicalindicators under salt and drought stresses; Molecular mechanisms analysis of tolerance salt onoverexpression GsZFP1and GsWRKY20by measuring Na+and K+content; Transgenic alfalfamarker genes expression by qRT-PCR; Analysis of stomatal size and density by scanning electronmicroscopy; Analysis of cuticle by transmission electron microscopy. These results would analyzemolecular mechanisms analysis of tolerance drought overexpression GsZFP1and GsWRKY20. Theresults of this study are as follows:1. Get3transgenic alfalfa with GsZFP1gene; Get3transgenic alfafla with GsWRKY20gene.2. GsZFP1and GsWRKY20genes enhanced transgenic plants tolerance salt, and the mechanism is：GsZFP1and GsWRKY20genes reduced absorbing Na+and discharging K+under NaCl treatmentand maintain the stability of sodium and potassium ion cells and protection of the intracellularenvironment stability and ion balance, thereby improving plants salt tolerance. 3. GsZFP1gene enhanced transgenic plants tolerance drought, and the mechanism is: Marker geneMtCOR47, MtRD2, MtP5CS and MtRAB18expression were upregulated under drought stress;Reducing stomatal size of alfalfa; increasing the thickness of the leaf cuticle and reducing the losswater of detached leaves into improving drought stress.4. GsWRKY20gene enhanced transgenic plants tolerance drought, and the mechanism is: Markergene MtCOR47, MtRAB18, MtP5CS, MtRD2and MtOST1expression were upregulated underdrought stress; Reducing stomatal density of alfalfa, increasing the thickness of the leaf cuticle andreducing the loss water of detached leaves into improving drought stress.