Cloning And Analysis Of The γ-gliadin TaWG05 Gene And Promoter From Wheat

Wheat as the world’s most widely cultivated food crops,has a very long history of cultivation,in China,more than half of the population to wheat as the staple food.With the continuous improvement of wheat breeding technology and field management,China’s total wheat production has been continuously increased income,the majority of people’s overall food and clothing problems have been resolved.However,in China’s grain market,the supply and demand of high-quality wheat is seriously imbalanced.Every year,China still needs to import high-quality gluten wheat from abroad to meet the demand of these people.The composition and type of wheat gluten are the most important factors affecting the quality of wheat processing,and the gliadin is the main component of wheat gluten.Therefore,the study of gliadin has very important theoretical and practical value.In the preliminary work,the seeds of high quality gluten wheat Zhengmai 366 and high quality weak gluten wheat Zhengmai 004 were analyzed by RNA-Seq(transcriptome sequencing)technique,and about 370 genes related to wheat quality were obtained.One of the Unigene expression in the two materials there was a significant difference,named Ta WG05.In this study,we cloned the gene and carried out its work around it,the following conclusions:1The full-length c DNA sequence of the gene was cloned by RACE technique and1、 The full-length cDNA sequence of the gene was cloned by RACE technique and cloned into 957 bp,encoding 319 amino acids.Analysis by Signal P software found that the N-terminal contains a 21 amino acid residues composed of signal peptide,suggesting that it belongs to the transmembrane protein.Using the Bio Edit software and the PROSITE database to predict its conserved domain,it was found that the protein had a typical γ-gliadin structure,and the result of phylogenetic tree analysis confirmed that the protein belongs to a new type of γ-gliadin.2、 The expression pattern of the gene was detected by real-time fluorescence quantitative PCR.The gene was only expressed in wheat seed.In order to further determine the specific expression site in the seed,the germplasm,embryo and endosperm were quantitatively analyzed,It was found that the gene was only expressed in wheat seed coat,and it was confirmed that Ta WG05 gene was a specific expression gene of wheat seed coat.3、 The promoter region of Ta GW05 gene was predicted by Plant CARE software,and the promoter-specific promoter elements GCN4-motif and Skn-1-motif were found in this region..4、 In order to further determine the expression pattern of this gene,we constructed the plant expression vector PMDC83-p Ta WG05-GUS,which was transferred into the commercial wheat cultivar Zhengmai 103 by gene gun method to obtain 16 positive plants.5、 GUS staining of the tissues at different developmental stages of transgenic plants revealed that only blue coloration was detected in the seeds,and it was found by paraffin partial thin section analysis that the coloring was present in the wheat seed coat and further confirmed that the promoter Express a promoter for a seed coat.