Cloning Of Germin Gene, Synthesizing Of Human Lysozyme Gene And Their Expression In Tobacco And Oilseed Rape

Qxalic acid is produced by several plant pathogenic fungi and is thought to have a primary role in the pathogenicity of several species, including the wide host-range pathogen Sclerotinia sclerotiorum. Infection by S. sclerotiorum causes serious damage to oilseed rape, soybean, sunflower and the mustard family vegetables. Because S. sclerotiorum is a non-host-selective pathogen, no germplasm resources among relative species of rape are found to be immune to the pathogen. Considering the importance of oxalic acid in pathogenicity, germin, an oxolate oxidase, was used in S. sclerotiorum resistance of crops breeding study. The wheat gernhin gene was cloned by PCR and a plant expression vector was constructed harboring the germin gene drived by CaMV 35S promoter. Germin gene has been transferred into the tabacco plants. Southern assay indicated that the target gene was integrated into tobacco genome. Enzymatic activity assay of oxalate oxidase in transgenic tobacco plants indicated that the germ in gene from monocotyledon, could be expressed in dicotyledon tobacco plants that resu;ted in the formation of homopentameric protein with enzymatic activity correctly. Cell wall hydralase has been used to degrade the cell wall of fungi and bacteria in plant disease resistance breeding. Human lysozyme(HL) is a bifunctional enzyme which displays strong lysozyme and chitinase activities. Thus the single foreign HL gene may confer the resistance to both fungi and bacteria pathogen in transgenic plants. In order to enhance the expression level of HL gene in plants, a human lysozyme gene was designed and synthesized according to the bias in codon usage by plant. The overall G-f-C content was increased from original 20.3% to 51.3%, and the G+C content at the third position was from 4 1.5% to 59.2% The potential processing sites such as AATTAAA and ATTTA which may result in the reduction of transcriptions translation and mRNA stability were avoided in our synthetic human lysozyme 3 gene. In order to show whether the synthesized HL gene was effective, a yeast expression vector was constructed. Western blot showed that the synthesized HL gene was expressed in yeast. Then a plant expression vector with HL gene and a-aniylase signal peptide, which would transport the I-IL into and enhance the accumulation level of HL in intercellular space, was constructed and used for tobacco transformation through Agrobacterium tumefaciens mediated gene transfer. Southern and Western blot assay indicated that the HL gene was integrated into and expressed in tobacco plants. In order to improve the resistance to S. sclerotiorum, a plant expression vector harboring both the germ in gene and HL gene was constructed and used for transformation of tobacco and oilseed rape. Western blot and enzymatic assay indicated that the HL gene and germ in gene was expressed in tobacco cell respectively. Dot blot assay showed that both germ in and HL genes were integrated into the genome of oilseed rape.