Genetic Engineering For The Enhancement Of Aluminum Tolerance In Medicago Sativa L.

Alfalfa (Medicago saliva L.) is one of the most highly valuable forage. Because of its advantage, it is widely used for livestock and poultry's food and to improve soil tilth. Alfalfa grows well in neuter or a little alkaline soil in northern China and the optimal pH range is 6 to 8. However, aluminum toxicity prevents the tranfer of alfalfa cultivation to the acid soil in the Long River region and south area of China. Generally, in order to relieve aluminum toxicity, people use lime to improve the soil pH.But this method can not solve the soil acid,because lime only can improve the top soil's pH.not improve the deeper soil.So people try to seek a new way to breed aluminum-tolerantce alfalfa.Presently, with the development of the transgenic technology, it is possible to improve the qualities of alfalfa by genetic engineering. To enhance the aluminum tolerance of this forage legume for the large-scale planting in southern China, this study conducted the following research work.Primers were designed according to the nucleotide sequence of citrate synthase (cs) gene. The endonuclease recognition site Nco I and Xba I were added to forward and reverse primer respectively. The cDNA of cs was amplified from tobacco total RNA via RT-PCR and then subclone into pMD18-T to yield pMD18-CS.To construct the light inducible plant expression vector, the pMD18-CS vector was digested with Nco I and Xba I and subcloned into the pUC-Rbcs-3c vector which contains the light induced promoter to replace the transit peptide and 3c gene fragments. The resultant plasmid pUC-Rbcs-cs was double-digested with Hindlll / Xba I and the target fragment was ligated into the HindIII / Xba I sites of a plant expression vector pPZP211 and generated a plant light inducible expression vector pPZP211-rbcs-cs.On the other hand, cs gene was amplified with Pryobest DNA polymerase by using pMD18-CS as a template. Blunt terminal PCR product was ligated into the gateway entry vector pENTR/D-TOPO. After identification by restriction enzymes and PCR, the correct entry clone, pENTR/D-TOPO-CS, was obtained. Gateway LR reaction was performed with pENTR/D-TOPO-CS and the plant expression vector pB2GW7 to produce a constitutive plant expression vector pB2-CS.Agrobacterium tumefaciens C58C1 (pPMP90) was transformed with pPZP211-rbcs-cs or pB2-CS. Alfalfa transformation was performed via Agrobacterium-mediated method. The leaves prepared from the highly regenetative sterile plantlets was infected and co-cultivated with the Agrobactertum tumefaciens. The callus was induced from the leaves but the transgenic Alafalfa plants are not obtained at present.At the same time, to obtain new strategy for genetic engineering in alfalfa for improvement of its Al-resistance, The cDNA was isolated for the genes which display different expression level in the treated and untreated Stylo (Stylosanthes spp) via different display PCR. Several DNA fragments...