Construction Of Highly Efficient Expression Vectors Of HMW-GS 1By15 And 1Dx1.5～t Genes For Transformation Of Wheat

High-molecular-weight glutenin subunits (HMW-GSs), one class of seed storage proteins, play an important role in determinating the bread-making quality of wheat flour. Recently, it had been found that the '1Bxl4+lByl5' subunits are positively correlated with the end use quality of wheat varieties. Interesting, the 1 B×l4+1 By 15 subunit pair is found to have a much higher influence on the sedimentation value of flour than the 'lBxl7+lByl8' subunits. Therefore, studying expression characters of Glu-1By15 will benefit for improving wheat quality via genetic transform.Proteins and mRNA were extracted from dry seeds and developing seeds of lBxl4+lByl5 carriers, Xiaoyan 54, Xiaoyan 6, Yanzhan 1 and Shanyou 225 to investigate the expression of the two genes at both transcription and translation leveles. Results indicated that the expression of Glu-1 Bxl 4 was much stronger than that of Glu-lByl5 at both levels and there is variability among varieties. Xiaoyan 54 and Shanyou 225 were utilized for further analyzing expression of lByl5 by semi-quantitative RT-PCR method. It was found that Glu-lByl5 only expressed in endosperm during a special developmental stage and the difference of expression stage and peak existed between the two varieties.Promoters of 2Bx2 4, 1By15, lBy8, 1Dx2 and 1Dy12 genes were cloned by PCR. Sequence analysis indicated that the HMW-GS genes were high homologous in their promoter regions. Some typical elements of HMW-GS promoter were found. Obvious difference was detected between 1Bx14 promoter and those of other HMW-GS genes. Transient expression showed that the promoter of 1By15 has lower activity than that of 1 Bx14, which was consistent with their transcription level. In addition, transient expression of the GUS driven by 1Dx2 promoter has the highest expression level.Two MARs sequences were cloned from tobacco and rice respectively. Transient expression experiment showed that it could not enhance the transcription of target genes after inserting a MARs before the expression cassette. Moreover, the expression of target genes was partially suppressed.1By15 and lDx1.5' gene expression cassettes were constructed, in which the two genes were controlled by the 1Dx2 gene's promoter. The expression cassettes were delivered into wheat by either particle bombardment or pollen tube method. After PCR analysis of To individuals, we found the transformation frequency of co-transformation plasmid pUC-MT2-15 and pACH20 was higher than pCAB2-15. After Southern hybridization, 4 T₀ individuals were confirmed being transformed by 1By15 gene. Six of them were confirmed to express the HMW-GS-lByl5 protein by SDS-PAGE.PCR analysis in the T₁ generation transformed by pCAB2-15 via pollen tube, 56 transgenic plants were identified. Two T₁ transgenic lines were confirmed containing lByl5 gene by Southern hybridization. Six T₂ seeds were confirmed to express HMW-GS 1By15 protein by SDS-PAGE. Western blotting analysis showed the unknown proteins in the LMW-GS of transgenic lines were not degradation of HMW glutenin subunit proteins.In transformation of 1Dx1.5' gene, PCR analysis in the T₀ population showed the transformation frequency of particle bombardment was about 1.9%.