AttM Transgenic Colored Calla Lily And Antibodies Preparation Of Ahl-Degrading Enzyme AttM

Colored calla lilies are monocotyledonous ornamental plants belonging to the family Araceae, which are described as "the star of flowers"in 21 century recently, the market prospect is good.But the bacterial soft rot disease, which caused by Pectobacterium carotovora subsp.carotovora,p.c.c maked the rot and death of colored calla lily in the field and storage period. Lacking of effective ways to control this disease, limited the development of colored calla lily seriously.The recent research found that there is a chemical communication between the different cells in the bacteria.When the chemical molecular reached a density range, the related genes experessed immeditly, which called quorum sensing. Recently, many quorum-quenching enzymes were found from procaryote and eucaryote, which could degrade the chemical molecular producing by the pathogen, interfere QS system, and reduce the virulence of pathogen to control the disease. Pectobacterium carotovora subsp. carotovora,p.c.c is gam-negative bacteria, which could produce N-acyl-homoserine lactone (AHL) molecular. More than ten kinds quorum-quenching enzymes have been found and reported. The sequences are different betweent different quorum-quenching enzymes. The quorum-quenching enzymes in the prokaryote belong to AiiA and AttM.In this research, attM gene was amplified from Agrobacterium tumefaciens by PCR using the specific primers designed based on the reported attM gene, and proper restriction enzyme sites were added at each end. It was cloned into pBI121 to make a recombinant vector pBI121-attM, which was proved to be constructed successfully by PCR, restriction enzyme digestion and sequence analysis. The resulting recombinant constructs pBI121-attM was transferred to E. coli DH5αby the freeze-thaw method. After verifying correct orientation of genes, this transgenic construct was transferred into Agribacterium tumefaciens EHA105. The positive clones were determined by PCR, restriction enzyme digestion and sequence analysis.The shoot basal discs from colored calla lily were co-cultivated with A. tumefaciens strain EHA105 carrying a plasmid pBI121-attM containing neomycin phosphotransferase, and 4 transgenic plants were obtained from about 10 000 shoot basal discs by screening with Kanamycin. Genomic integration and expression of the transferred genes were determined by gus analysis and PCR.In order to do more study and use of attM gene to control the bacterial soft rot disease, attM gene were amplified from the Agrobacterium tumefaciens by PCR using the specific primers designed based on the reported attM gene. They were separately transplanted into vector pET-30a to construct expression vectors named pET30a-attM, and expressed in host strain BL21. The result from SDS-PAGE showed that the attM gene were successfully expressed AHL-degrading enzyme AttM respectivly. The SDS-PAGE products of the attM gene were used directely to immunize adult rabbits. Both ELISA and Western Blot all demonstrated that such way for AHLase testing were quickly and efficienely.