Transformation And Transgenic Medicago Sativa L Identification Of The Glycerol-3-phosphate Dehydrogenase Gene From Dunaliella Salina

Drought and salination are the key factor of environmental crisis. So the research on the halo-tolerant gene engineering will play extreme important function for the alteration to the salination of soil. Dunaliella salina is the most extreme halotolerat alga in the world , The eukaryotic unicellular green alga able to proliferate under saline condition ranging from 0.1 to 5.5 mol/L NaCl. It is a good model organism in the plant halotolerance research. The alga regulates its inside turgor to adapt the outside osmotic change by synthesizing a large amount of glycerol. It is important to study glycerol-3-phosphate dehydrogenase gene, the key enzyme gene of the glycerol metabolism to know how the species can sustain the hyperosmotic press by accumulating so much glycerol. We introduced DsGPD gene into Medicago sativa L by gene engineering and take the hereditary change of the alfalfa halo-tolerance into practice, it can enlarge the growth range of alfalfa, which play important role in improvement environment, prevention the loss of water and soil and enhancement the nutrition of soil.We have got the DsGPD gene from the cDNA library. Now, we constructed the vector pCAMBIA2301G-DsGPD, which was introduced into Agrobacterium tumefaciens EH105 using the freeze-thaw method. After infected by A. tumefaciens, the explants of Medicago sativa L were selected on platescontaining UM inducement medium supplemented with 50mg/L kanamycin. Soon, calli were inducted and form embryoid, after three month, the embryoid germinate .We cut the regeneration bud and insert into MS3 medium .The rootage rate of regenerated plants was 60%. Regenerated plants transplanted into vermiculite: sand (1:1) medium. We got regenerated plants 102 strains, distill the total DNA of regenerated plants as the template of PCR, PCR analysis showed that approximate 21% tested plants produced the target band. We constitute the system to transformation and regeneration, set up the basis for the further function search and enhance the salt tolerance of Medicago sativa L.Compare the efficiency of transform, cotyledon and lamina were transformed more easy than hypocotyls. When the OD value of Agrobacterium tumefaciens was 0.3 ~0.5 ,the rate of callue formation was 60%~80%.The rate of differentiation was 17%.We transfer DsGPD to alfalfa lamina under the circumstance of ultrasonic and vacuum. GUS stain to the lamina and callus, then we get the efficiency of gene-transferring. The rate of transformation under the circumstance of ultrasonic and vacuum was 27.7% and 22.2% respectively, it was enhanced clearly under the circumstance of ultrasonic and vacuum. In addition, DsGPD was transformed into the callus of medicago, the best condition of electric-transformation was voltage 650V/cm, capacitance 50 uF which was the best condition to transform the Medicago sativa L.