Transformation Of Peste Des Petits Ruminants Virus With H,F Gene Into Alfalfa By Agrobacterium-mediated

The H gene of peste des petits ruminants virus (PPRV) was transferred to alfalfa by Agrobacterium-mediated transformation which would lay the foundations for the preparation of transgenic forage vaccine that may be prevent and cure PPR.To construct the plant expression vectors of PPRV, the gene of H and F from PPRV plasmid, H-KDEL through adding KDEL into the gene H before its termination codon, two sequences MAR12 and MAR34 from Tobacco SRI which could enhance the expression of exogenous genes, as well as a reporter gene, green fluorescent protein gene from pHL005 plasmid, were cloned by TA cloning. All the clones were with the enzyme site. The results showed that the clones size of all the genes and sequences were the same to the theories. The cloning vectors had been constructed successfully. The GFP gene was inserted into pBI121 in order to replace the GUS gene, and the tissue can be detected easily. PPRV-H gene and PPRV-H-KDEL gene were inserted into pBI121-GFP, then the plant expression vectors pBI121-GFP-H and pBI121-GFP-H-KDEL were constructed as well. MAR sequence was inserted into the side of pBI121-GFP, the middle expression vector MAR-pBI121-GFP was constructed, then PPRV-H gene, PPRV-H-KDEL gene and PPRV-F gene were inserted into MAR-pBI121-GFP, the plant expression vectors MAR-pBI121-GFP-H, MAR-pBI121-GFP-H-KDEL and MAR-pBI121-GFP-F were constructed. The plant expression vectors were testified by enzyme cutting and PCR amplification, and the results were right.The expression vector pBI121-GFP-H was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method using the hypocotyls of alfalfa cultivar Gan-nong No.1. as explants after the plant expression vectors were constructed. Further transformation system was optimized:the hypocotyl was immersed in the bacterial suspension for 15 minutes after pre-cultivated for 3 days, then co-cultivated on MS medium covered with a piece of sterile filter paper for 3 days, the density of Km and Cef were confirmed as 75mg/L and as 300mg/L, repectively. The Km-resistant transformants were obtained by Km screening while no resistant plant was gained. Green fluorescent was not detected by the ultraviolet lamp. The positive callus were not gained by PCR amplified from resistant callus DNA. and this should be researched further in the future.