Expression Analysis of HMW-GS 1 Bx14 and 1 By15 in Wheat Varieties and Transgenic Research of 1By15 Gene

摘要

High-molecular-weight glutenin subunits (HMW-GSs), one class of seed storage proteins in wheat, play an important role in determining bread-making quality of flour. More and more proves support that HMW-GS-1Bx 14 and -1Bx15 subunits are strongly positively associated with good bread-making and excellent noodle-making quality. The two subunits are encoded by two genes, Glu-1Bx14 and Glu-1Bx15, which are tightly linked and located on the 1BL. Protein assay by SDS-PAGE indicated that the expression of Glu-1Bx14 gene was always much stronger than that of Glu-1By15 in the same variety. But, variation of expression level for Glu-1By15 gene existed among varieties, such as in Xiaoyan 54, Xiaoyan 6, Yanzhan 1 and Shanyou 225. We also investigated the transcription difference of Glu-1By14 and Glu-1By15 genes in Xiaoyan 54 and Shanyou 225 by semi-quantitative RT-PCR method. The Glu-lByl4 always transcripts much more than the Glu-1By15. This was basically consistent with the translation difference between thetwo genes. Promoters of 1Bx14, 1By15, 1By8, 1Dx2 and 1Dy12 were cloned from Xiaoyan 54, Chinese Spring and Aegilops tauschii. Sequence analysis indicated that the HMW-GS genes had high homology at their promoter regions. However, significant differenceexisted between sequence of 1Bx14 promoter and those of other HMW-GS genes. The transient expression experiment showed that the promoter of IBy15 has lower activity than that of 1Bx14, which was consistent with their transcription level of the two genesin varieties. In addition, transient expression of the gus driven by the promoter (P2) of HMW-GS 1Dx2 gene was higher than by other HMW-GS promoters. Therefore, we constructed 1By15 gene expression vector driven by the 1Dx2 promoter, and transformed the1By15 gene into wheat commercial variety, Jimai 20 by pollen tube method. Of 45 independent transgenic lines identified by PCR, 3 were confirmed to contain the HMW-GS 1By15 gene via Southern hybridization. The delivered 1By15 gene expressed the expectedHMW-GS protein in the seeds of transgenic plants.