Cloning And Characterization Of Monodehydroascorbate Reductase Gene MsMDAR In Medicago Sativa L.

Based on the homology of the gene in different species, a section of cDNA wascloned from alfalfa. After homology analysis in NCBI, it is suggested that this sequenceencodes monodehydroascorbate reductase, marked as MsMDAR. The coding region ofthis gene was1302bp in length, encoding434amino acids. Its Genebank accessionnumber is JN979555in NCBI.The ORF (open reading frame) of MsMDAR was introduced into prokaryoticexpression vector pET-30a(+), and the fusion expression vector pET-MDAR wasobtained. This fusion vector was introduced into E.coli BL21(DE3) and induced byIPTG, the induced product was a fusion protein with His-tag. This protein expressed inE.coli efficiently and specifically by SDS-PAGE analysis, and the product size wasconsistent with the prediction. The open reading frame of MsMDAR was introduced intothe expression vector pA7-GFP, and the fusion expression vector GFP-MDAR wasobtained. The fusion vector was introduced into onion epidermal cells by gene guntechnology and the fluorescence intensity distribution was observed under confocalmicroscope. The results showed the protein located in nucleus and cytoplasm of onionepidermal cells.The open reading frame of MsMDAR was introduced into the expression vectorpBI121, and the fusion expression vector pBI-MDAR was obtained. The fusion vectorwas transferred to Agrobacterium tumefaciens GV3101by freeze-thaw method, and thepositive agrobacterium was transferred to tobacco by agrobacterium-mediated method,expecting to gain MsMDAR over-expression tobacco. Regenerated plants were detectedby PCR, RT-PCR and GUS staining and finally only two transgenic seedlings wereobtained. MsMDAR gene RNAi vector was obtained though pHANNIBAL intermediatevector and pART27expression vector, and then was transferred to alfafla byagrobacterium-mediated method, hoping to get MsMDAR low-expression plant.Regenerated plants were detected by PCR, showing that the expression of MsMDARwas interfered in80%of the regenerated alfalfa.