Overexpression Of Wheat Prolamin-box Binding Factor Gene TaPBF-D In Wheat Promotes The Accumulation Of Seed Glutenin

Wheat is one of the three important cereal crops in the world. It can be processed into various types of foods because its seeds are rich in prolamin, which is composed of gliadin and glutenin. The latter is is divided into high-molecular-weight glutenin subunits (HMW-GSs) and low-molecular-weight glutenin subunits (LMW-GSs) based on the molecular weight, which determined the unique viscoelastic properties of flour. Although it has been reported that the expression of glutenin was mainly controlled at transcriptional level, there is no study on wheat transformation by TaPBF-D.The full-length cDNA coding sequence of TaPBF-D which has been cloned in our previous study was amplified and inserted into pGA3626 expression vector, and then integrated into JM22 genome by the wheat shoot tip transformation technique. Ten To, four Ti and two T2 positive wheat transgenic lines were confirmed by PCR and qRT-PCR. The results of qRT-PCR indicated that the expression levels of 1 By8,1Bx7 and lDy12 were increased in the wheat TaPBF-D overexpression (OE) lines than those in the wild. It was also found that the expression level of the endosperm-specific transcription factor (TF) TaSPA was increased significantly, while another related TF TaGAMYB was not. The correlations between related TFs and glutenin subunits were determined by software SPSS2.0. It was indicated that the overexpression of TaPBF have remarkable positive correlations with the expressions of TaSPA, lBy8,1Bx7 and 1Dy12. Besides, seed glutenin content of Ti and T2 in positive wheat transgenic lines also increased apparently compared to those in control using protein quantitative analysis. It was concluded from the above results that the enhanced expression level of TaPBF-D can regulated the expression of 1By8,1Bx7 and 1Dy12 at transcriptional level with direct action on corresponding cis-element and undirect interaction with the other related TF, such as TaSPA. In order to investigated whether the change of transcriptional level influenced epigenetic regulation, we detected the expression levels of the DNA methylases (MET2a, MET2b, MET3, CMT, Dnmt and DRM) and demethylases (DME, DML) which expressed in endosperm and found that there were no regular expression changes of the DNA methylases and demethylases in wheat TaPBF-D OE lines.Using the wheat HMW-GS mutants, the expression levels of related TFs TaPBFs(-A,-B,-D), TaSPAs(-A,-B,-D), TaGAMYB, the DNA methylases (MET2a, MET2b, MET3, CMT, Dnmt and DRM) and demethylases (DME, DML) were also detected in different endosperm development stages. The result indicated the expression levels of the TFs TaPBFs, TaSPAs and TaGAMYB was the highest in the wild type wheat L03-227 (HMW-GS:5+10,1,17+18) and the lowest in the wheat mutant L03-221 (HMW-GS:Null) respectively. The expression levels of TaPBFs and TaSPAs reduced gradually with the number of glutenin subunit lost increasing in other wheat mutants. The expression of TaGAMYB had no regular change compared to TaPBFs and TaSPAs. The expression levels of the DNA methylases and demethylases had no regular change. These results suggested that the expression of HMW-GS were associated with the expression of TFs TaPBF and TaSPA.Finally, the coding sequences of PBF from wheat cv. SR3 were cloned. By sequence alignment, it was found that the cDNA sequences of PBF in wheat introgression lines SR3 were highly homologous with those in its parent JN177. The results showed that the gene sequences of PBF in SR3 were derived from its receptor parent JN177 rather than its donor parent tall wheat grass.