Mechanisms For Differential Expression Of TaWx-1 Homologous Genes In Common Wheat

Starch is the main component of wheat grain,which accounts for 65-75% of its dry weight and is an energy source for human beings.Granule-bound starch synthase(GBSSI)is a key enzyme in wheat amylose synthesis and is closely related to amylose content.GBSSI is encoded by TaWx-1 gene.In common wheat,TaWx-1 loci consist of three homologous genes,TaWx-A1,TaWx-B1 and TaWx-D1.In this study,we verified the universality of the differential expression of TaWx-A1,TaWx-B1,TaWx-D1 at the transcript level and enzyme activity level.The effects of coding region and promoter region of TaWx-A1,TaWx-B1,TaWx-D1 to the gene expression were analyzed preliminarily.The molecular mechanism of differential expression of TaWx-1gene was preliminarily revealed through functional studies.The main findings are as follows:1.The differential expression among TaWx-A1,TaWx-B1 and TaWx-D1 was verified at gene expression level and enzyme activity level.At different stages of seed devolopment(5,10,15,20,25,30 days after flowering,DAF)of Shumai 830(SM830),Shumai 969(SM969)and Shumai 482(SM482),the gene expression of TaWx-B1 was higher than that of TaWx-D1 and TaWx-A1.The near isogenic lines with different Wx protein null types of SM969 were used to measure the enzyme activity of GBSSI.The results showed that the GBSSI enzyme activity of TaWx-B1 was the highest among three alleles,followed by TaWx-D1,and the lowest was TaWx-A1 which was consistent with the results of previous studies.2.By analyzing the gene structure and function of TaWx-A1,TaWx-B1 and TaWx-D1,it was determined that the difference of promoter elements was the reason that affected the expression of TaWx-1 gene.The difference in coding region is small,which is not the reason for the difference in expression.The type and content of cis-acting elements in TaWx-1promoter region predicted by plant CARE were significantly different and the promoter activity was TaWx-B1> TaWx-D1 > TaWx-A1.When the promoter of TaWx-B1 was reduced from2500 bp to 2000 bp,the promoter activity decreased sharply.Therefore,-2500 bp to-2000 bp was identified as the key action region.Two cis-acting elements CRTDREHVCBF2 and IBOX were identified in this region,which affected the activity of TaWx-B1 promoter.A candidate transcription factor BRX1 that may interact with IBOX was identified by screening of yeast library.3.TaWx-A1,TaWx-B1,TaWx-D1 genes were overexpressed in barley,but the alien wheat Wx protein was not able to bind to the starch granules of barley.The complete expression cassettes of TaWx-A1,TaWx-B1 and TaWx-D1 were characterized and constructed in p CAMBIA1302 vector.The transgenic plants were obtained by Agrobacterium-mediated transformation,and the T1 generation homozygous plants were obtained in greenhouse.The expression levels of TaWx-A1,TaWx-B1,TaWx-D1 genes were increased in transgenic plants.The Wx protein in transgenic plants was identified by SDS-PAGE and Western blotting,the results showed that only native Wx protein of barley was detected,but not in the starch granules.The amylose content of transgenic plants was not different from that of wild type of barley.PTST protein can mediate the correct localization of Wx protein on starch granules by interacting with Wx protein through coiled helix domain.There are amino acid differences between the PTST binding domains of wheat Wx protein and barley Wx protein.The results of Yeast two-hybrid and BIFC indicate that the interaction between wheat Wx protein and barley PTST protein is weak,resulting in the failure of barley PTST protein to correctly locate the transferred wheat Wx protein to barley starch granules.