TaSNX1 Gene Function Analysis For Wheat And Low LOX Activity Genetic Improvement Of Wheat

Phosphorus has an irreplaceable role on the growth and development of crops.The lack of Phosphorus in the soil is great threat to yield of wheat.Consequently,finding the mechanisms of wheat to low phosphorus stress resistance and screening wheat genotypes with high phosphorus use efficiency are increasingly important to maintain wheat yields.Fatty acid oxidase（LOX）has a great impact on the quality of wheat.Study on creating novel wheat germ plasm with good application prospect by using genetic engineering to reduce LOX activity has an important significance to ensure wheat quality.In this study,low phosphorus stress gene TaSNX1 of wheat was cloned and studied the preliminary functional analysis.Field inspection and the identification of molecular identification and enzyme activity have been conducted on constructed Low LOX activity RNA i transgenic wheat.Major conclusions were as follows:1.TaSNX1 is the important screened gene involved in phosphorus stress response.The results of functional analysis of TaSNXI are as follows:（1）Cloning and Sequencing of TaSNXlTaSNX1 contains 10 exons and 9 introns within the genomic DNA sequence and has one open reading frame of 1203bp encoding a protein with 400 amino acids.Single nucleotide polymorphism（SNP）analysis found that 28 amino acid mutations cause changes in five amino acids in the cloning of Zhengmai 9023.（2）Constructing the TaSNX1 transgenic ArabidopsisBy using the vector p1 300 recombined from strong promoter masl and green fluorescent protein（GFP）which can specific express in plant root owns,AtSNX1 over-expressing transgenic Arabidopsis was constructed and then a positive mutants was obtained through resistance screening and PCR amplification.（3）Prokaryotic expression analysis of TaSNXl geneProkaryotic expression vector pET-32a-TaSNXl was construted and TaSNXl fusion protein was successfully obtained.TaSNX1 encodes 400 amino acids whose molecular weight is 46.0 KD.Coexpression vector pET₃2a-TaSNX1 encodes a soluble protein containing 551 amino acids and the protein molecular weight is 61 KD.2.Results of study on transgenic wheat with low LOX activity:（1）molecular screening wheat and physiological shape analysis of low LOX transgenic wheatBy selfing transgenic wheat with a marker gene Bar and target gene can produce offspring containing only the target gene.By screening we obtain 20 transgenic wheat lines containing only target gene and 51 transgenic wheat lines which have marker and target gene in the four transgenic wheat varieties.By analysis of Transgenic wheat physiological shape like plant height,grain weight and spike,we find that transgenic wheat is generally lower than normal wheat.（2）The analysis of LOX activity in transgenic wheat grainAnalysis of LOX activity by spectrophotometer method found that transgenic wheat including Zhoumai 18,Yunong 202,Yumai 57,Aikang 58 have the decreased LOX activity when compared to non-transgenic wheat transgenic wheat.Among the transgenic wheat,Zhoumai 18 has the highest LOX activity and Yunong202 has the lowest LOX activity..