Cloning Of MsCS Gene From Alfalfa (Medicago Sativa L.) And Transformation Of Tobaccos

The citrate synthase which is one of the active enzyme can regulate the organic acid in the plant,and it is a key enzyme which is concerned with oxaloacetic acid and acetyl-CoA to formed the citric acid. Alfalfa is one of legume forages,and it have planting area about more than 30 million hm2. China is one of the country which used and planted more alfalfa of the world. There are 14 provinces and areas to plant the alfalfa ,and mostly in north, northwest. But in the south , alfalfa planting is quite limited, someone think that the main factor is the Al³⁺ in the acidic soil. By the genetic transformation method will take the citric acid synthase gene transformation into the plant which is sensitive about the aluminum, that can improve the ability of the plant about the al-tolerance, and is the best way to slove the aluminum toxicity in the plant.A full-length cDNA,named MsCS,encoding a citrate synthase was cloned from alfalfa (Medicago sativa L.) by rapid amplification of cDNA ends(RACE).The length of cDNA sequence was 2031bp and included a 1551bp open reading frame which encoding a deduced protein of 517-amino-acid polypeptide. The amino acid sequence compared by blast and DNAman revealed high homology with citrate synthase of other plants,like Glycine max, Arabidopsis thaliana, Oryza sativa Indica Group, Sorghum bicolor.Plant expression vector named pBI-MsCS was constructed based on pBI121 vector.Using agrobacterium-mediated to transform the gene into the tobacco, that we obtained 17 kanamycin resistant transgenic tobacco plants. And the PCR detection confirmed 6 positive plants.