Variations Analysis Of Wheat Storage Protein From High Pressure

In order to verify the effectiveness of high-pressure mutagenesis on the wheat qualityimprovement, storage protein genetic variation of39wheat pressure mutagenesis offspringwere investigated, through the use of polyacrylamide gel electrophoresis and capillaryelectrophoresis analysis. Protein points of difference were identified by mass spectrometry,through the use of MALDI-TOF-MS. Meanwhile, Physical, chemical quality and doughrheological properties of the wheat pressure mutagenesis offspring were tested. Theseexperiments are trying to find the relationship between the variation of protein and qualitytraits, prove the effect of wheat high pressure mutagenesis and provide new means for plantgermplasm.The experimental results are as follows.(1)The relative content of different types of subunit can be clearly distinguished in theglutenin HPCE map. If exclude the acetone precipitation loss in protein extraction, content ofeach subunit in different wheat varieties can be accurately calculated. So compared withtraditional methods, glutenin HPCE map is more accurate, reliable and simple. There aresignificant differences between HMW-GS and LMW-GS peak time. HMW-GS peak time isbefore25min, while LMW-GS peak time is after25min, and this result is coincides withmolecular weight difference. From the separation effect of LMW-GS, method established inthis study can distinguish most of the LMW-GS, which obviously better than the SDS-PAGE.At the same time, the better HMW-GS removed, the more obvious LMW-GS separation; TheSDS-PAGE and capillary electrophoresis analysis of HMW-GS and LMW-GS show that,whole HMW-GS of High-pressure mutagenesis offspring did not change significantly, butLMW-GS changed slightly. And its effects on the quality needs further study.(2)Gliadin loci varied richly when wheat was mutagenized through high pressure, whichfully proved high pressure mutagenesis is an effective breeding method; The variation ofgliadin bands can be divided into four categories. Variation occurs mainly in ω and γ district,at the same time, there were difference of the expression intensity of the β district, howevervariation occurs rarely in α and β district.Further, compared with A-PAGE, there are obviousadvantages for capillary electrophoresis to analyze the closer relationship wheat gliadin. The best gliadin HPCE separation condition in this paper is: phosphoric acid-glycine buffer (20%acetonitrile,0.05%Hydroxypropyl methyl cellulose and pH2.5),50μm inner diametercapillary column, Injection8sec in10KV voltage, continuous separation50min in18KVvoltage and40℃;30~35peaks can be separated from the sample between10~50min, andthe separable degree is significant higher than the A-PAGE. The variation of gliadin can bedivided into four types, which consist with A-PAGE results, and the main change is the peaknumber and area in15minutes and between30and35minutes.(3)It was a basis for judgment, protein staining density changing in two times. GliadinA-PAGE protein was analyzed, and six spots changed significantly. These changed proteinswere analyzed by MALDI-TOF MS mass spectrometry, searched in NCBI non-redundantprotein database through Mascot. But only four protein spots were identified effectively,which may be related to the gliadin information missing. Identification rate of66.7%meetsthe usual identification of test dye rate. The analysis of MALDI-TOF MS mass spectrometryshows that, the seed storage protein is gliadin concentrated in1500-4500kD. But thecomponent usually concentrated between1500~4500kD, which difficult to separate. Whilethe smaller molecular weight protein in the map likely is albumins, globulin or otherimpurities. Gliadin is extracted with70%ethanol, inevitably with a water-soluble protein. Themolecular weight of ω gliadin is large, about3500~5000kD. Content of ω gliadin is relativelylow (about15%of gliadin), scattered protein peaks between4500~8000kD in the map.(4)Gliadin changed when wheat was mutagenized through high pressure. Therefore,simple correlation analysis of quality traits of changed bars was investigated,show that thecorrelations between the6mutation bands4and the8wheat quality characters weresignificants or extremLy. The overall trend is total gliadin bands decreases, but some qualityof the dough improve, when wheat was mutagenized through high pressure.