Molecular Characterization Of Three Types Of Storage Protein Transcription Factors And Two Types Of Methylase Genes From Wheat

Total27full-length cDNA sequences of three types of transcription factors (SPA, PBF, GAMYB)were isolated from unmatured seed endosperms of three different wheat cultivars (JN177, SR3and Chinese Spring), using homologous gene cloning technology and RT-PCR method. Based on sequence clustering and comparing, we found that each type of transcription factors has a high homologous sequence in A,B3D sub-genome of three wheat cultivars, and the similarity level varied from95.73%to98.6%. It indicated that those homolog genes characterized by different SNPs are conserved in evolution.In addition, we isolated a GAMYB-like gene from tall wheatgrass leaf. We found GAMYB-A of SR3is highly homologous with GAMYB-A of JN177and GAMYB-D of SR3is highly similarity to GAMYB-D by comparison of GAMYB genes from hybrid SR3and its parents JN177and tall wheatgrass.Semi-quantitative and quantitative real-time PCR were used to analyze expression levels of three transcription factors in different stages of developmental seeds from wheat cultivars SR3, JN177and Chinese Spring.The expression levels present in SR3were significant higher than those in JN177and Chinese spring. Meanwhile, we found that both of SPA and PBF expressed in nuclear and GAMYB expressed in both nuclear and cytoplasm according to Subcellular location. One gene of each type from SR3was chosen to transfer into wheat based on gene expression levels. Total15positive wheat plants were selected after PCR analysis. The transformed wheat will be used in futural research and those results together will" be helpful to understand how those transcription factors regulate gluten expression.Using electronic cloning method, five methylase genes which belong to four types by Phelogenetic analysis were spliced. Two full-length genes were cloned from model wheatusing a genomic PCR strategy with sequence-specific primers, the one is DRM-like gene, another one is Dmnt2-like. Besides, TaDRM expressed in both nuclear and membrane according to subcellular location. Two positive transformed TaDRM wheatplants were obtained using shoot tip transformation technique, which will further be used to study the relationship between promoter methylation and gene expression of glutenin genes.