| Wheat is one of the most important crops in the world, and the plant area of wheat is about 30% of all crops. The flour can be machininged all kinds of foodstuff and it is one of the sources of proteins for mankind. Therefore on the study of wheat storage protein is one of the focuses in cereal science.High molecular weight glutenin (HMW-GS) is one kind of the most important storage proteins of wheat. It is reported that the quality of wheat was greatly affected by HMW-GS and some certain HMW-GS may improve the quality of the wheat. Therefore, the isolation and purification of single HMW-GS and function study of the HMW-GS are very important in wheat breeding.SDS-PAGE, RP-HPLC and HPCE were used for isolation and purification of HMW-GS. But each of these methods has its advantage and disadvantage. The objective of this study was to establish a new method for isolation and purification of HMW-GSs by one-step FPLC. Four wheat varieties with different HMW-GS were selected as materials (Xiaoyan6 xiaoyan503 Yuzhan2000 Jinan17). The following results were obtained:1 The optimal elution condition of FPLC was the gradient formed with buffer A (0.05M Tris-HCl containing 4M urea and 0.25M (NH4)2SO4, pH8.0) and bufferB (0.05M Tris-HCl containing 4M urea pH 8.0) at a flow rate of 0.5ml/min.2 1Dx2 subunit and 1Bx14 subunit can be purified from Xiaoyan6, 1Bx7 subunit and Dx2 subunit can be got from Xiaoyan503, 1Bx13 subunit and 1Dx4 subunit can be got from Yuzhan2000, 1Bx7 subunit and 1Dx5 subunit can be got from Jinan17 by FPLC respectively. The order of elution was, y- type subunit, 1Dx subunit,1Bx subunit,1Ax subunit.3 1Bx and 1Dx-type HMW-GS or all y-type HMW-GSs in any genotype can be isolated and purified with one-step FPLC developed in the present study.4 HMW-GS1Bx14 was purified by Reaction Red-120 affinity chromatography and the optimal elution condition was that 0.05M Tris-HCL+ 4M urea (pH8.0) + 0.04% SDS.These results offer new methods to purify the single HMW-GS. |
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