Research Of Transgenic Wheat Mutant Protein And Reliminary Mapping Of1Dx5Gene

Safety of genetically modified food is one of the world’s problems to be solved.Although the transgenic technology has been successful, but with the introduction offoreign genes, unknown protein and gene silencing appeared, which result from thechange of different degrees of genome. The reason many generations of genomicmutations, one of the important reasons is that the integration of the exogenous genelocus. The integration site of exogenous gene is random integration or site directedintegration is still controversial although researchers generally believe that foreigngene integrated in the priority autosomal enrichment gene zone. This research willstudy the mutant protein1Dx5gene sequence and external source location to exploreintegration of exogenous gene in wheat genome rules, so as to provide theoreticalbasis for the study of transgenic wheat. At the same time, this study established anew separation purification system, optimized the purification parameters to obtainprotein powder with high purity and then obtain the unknown protein sequences, andlay the foundation for obtaining its coding gene.The research obtained results are as follows：The finding of high molecular weight mutain protein.SDS-PAGE was detectedby16kinds of mutants found3mutations, B72-8-11b of transgenic wheat B73-6-1,3mutants of B73-6-1have been mutated protein a size of about120kD (1Ax1proteinabove), fifth kinds of mutant B72-8-11b also appeared a protein of the same size.The establishment of a new protein separation and purification system, optimizethe purification part parameters.Protein subunit recovery of existing purificationmethod was improved, mainly analyzed from SDS-PAGE gel dyeing bleaching time,sensitivity and electro elution buffer and protein recovery angle, and expounds themechanism of dyeing, electric elution. The protein recovery efficiency is improved5times more than the traditional method, and the recovery time is reduced greatly,need only about20min dyeing and decolorizing time, quite at the same time,sensitivity and traditional methods, or even higher than that of the traditional method.The mutant protein sequence.Separation and purification of protein powder ispure system using new protein, sent to the company for sequencing, N endsequencing result showed that, the protein N terminal five amino acid sequence(EGEAS) and partial amino acid sequences with almost all HMW-GS has homology,100%at the same time, the results showed the protein identification by massspectrometry, the two highest scoring (96and58) peptides (GGSFYPGETTPPQQLQQR and IFWGIPALLKR) and1Dx5subunit homologyreached100%. Preliminary evidence, the mutant protein of high molecular weightglutenin subunits new. Provide more basis to study the exogenous1Dx5geneintegration site determination and the exogenous gene integration rule.To obtain the location of1Dx5gene by using fluorescence in situhybridization.In order to verify the position relationship within, exogenous1Dx5gene and gene mutation, the study of1Dx5gene by fluorescence in situ hybridization,after an initial detection, two fluorescent bright spot appeared in the receptor wheatL88-6and wheat1Dx5gene B73-6-1D group1chromosomes, and only in this pairof homologous the chromosome appears bright spot. It can be speculated, exogenous1Dx5gene might have a site-specific integration, integration of exogenous1Dx5gene may be due to endogenous1Dx5gene or internal, not destroy the normalexpression of the endogenous1Dx5gene, but also greatly increased the expression ofthe bread wheat genes, and may use the same promoter and expression of amolecular weight far greater than mutations in the gene1Dx5, resulting in highmolecular weight protein production, the test will be the following.