Transformation Of E.coli Malate Drhydrogenase Gene Into Medicago Sativa L. Mediated By Agrobacterium Tumefaciens

Alfalfa(Medicago sativa L.) is a perennial leguminious forage.It grows well in the warm,sub-humid and semi-arid area,Alfalfa grows mainly in Yellow River area and the northwest in our country.In south,Alfalfa's plant is restricted in acid soil.The poison of aluminum to alfalfa is a key problem in acid soil.So,The enhancement of aluminum tolerance in Alfalfa is a key to introduce alfalfa in the south.In recent years,The development of molecular biology technology and mechanism research of aluminum tolerance,especially enhancing organic acid synthesis in plants may be an effective strategy to cope with soil acidity and A1 toxicity,it is possible to improve alfalfa to aluminum tolerance by genetic engineering.This research intended to transfer the E.coli Malate Drhydrogenase Gene(EMDH) into Alfalfa by Agrobacterium -mediated transformation.The expression of the epigenetic gene could strengthen the synthesis and exudation of malate in alfalfa accordingly.Exudation of malate from root of alfalfa would relieve aluminum toxicity in acid soil.There had acquired main results as follows:Determination of the appropriate antibiotic concentration.Prophase filtration to Hyg as appropriate way was determined by examination.Besides,we determined 5 mg/L Hyg as screening stress in callus inducement phase and differentiation phase.We also determined 500mg/L Car as Sterilization culture in callus inducement phase and differentiation phase respectively.Obtain of genetic transformation plant.After filtrated culture,29 resistance lines were obtained. It was proved by PCR examination that target gene MDH had been transferred into alfalfa.One positive line was obtained.It was proved further by RT-PCR examination that the expression of EMDH gene in transcription Level.Enzyme activity analysis showed that the MDH activity of transgenic line was increased by 16%than unransgenic line.In normol case,Oraganic acid was increased by 1.6 fold compared with control;In the solutions containing 50μmol/L Al³⁺,Oraganic acid content in leaf of transgenic plant was 3.4 fold more than control.For tolerance examination,it was showed that the transgenic plant can tolerate aluminum toxicity.In the solutions containing 20μmol/L Al³⁺,Relative root growth of transgenic plants was more than untransgenic plant.