Study Of Efficient Separation And Identification Of High Molecular Weight Glutenin Subunits By HPCE

High molecular weight glutenin subunit（HMW-GS）is an important factor determining wheat baking quality.The composition of HMW-GS is closely related to the quality of Wheat.The conventional methods used for seperation and identification of HMW-GS mainly include SDS-PAGE,molecular markers and RP-HPLC,which have varying degrees of limitation.High performance capillary electrophoresis（HPCE）has a good application prospect in separation and identification because of its less sample,faster speed and higher resolution.But,at present,the researches of HPCE for HMW-GS on wheat are still in its infancy,there are few reports about the standard spectrum,and the separation efficiency and resolution of existing separation systems are still need to be improved.Based on this,twelve wheat varieties were selected to study the HPCE high efficiency separation system and spectrum of HMW-GS with IDA buffer system.The result is as follow:1.The results of SDS-PAGE and molecular marker analysis showed that the tested varieties had diversity in HMW-GS composition.Their subunit types include 1Ax1、1Ax2*、1Dx2、1Dx3、1Dx4、1Dx5、1Bx6、1Bx7、1By8、1By9、1Dy10、1Dy12、1Bx13、1By16、1Bx17、1By18、1Bx20和1By20.2."Chinese Spring"wheat was selected as a standard sample.The effects of buffer system and electrophoresis parameters on the separation of HMW-GS were systematically studied.The high efficient separation system of HPCE was established by optimizing above conditions.Buffer components:15%ACN+0.05%HPMC+75 mmol L^-1 IDA,pH 2.5;electrophoresis parameters:operating temperature is 30℃,separation voltage is 20 kV,capillary inner diameter is 25μm,detection wavelength is 200 nm,and simpling time is 5 s.The results of continuous repeat tests and comparison analysis showed that the established system has good separation efficiency and reproducibility.3.3.Based on the established system,the HPCE spectrums of different subunits were studied by the method of mixed injection with controlled variable.The standard spectrums of 18 subunits were obtained,their migration sequence are:1Dy12→1Dy1→1By9→1By8→1By18→1By16→1By20→1Bx17→1Bx20→1Bx13→1Bx6→1Bx7→1Ax2*→1Ax1→1Dx5→1Dx4→1Dx3→1Dx2,their standard migration time are:9.39、9.69、10.30、11.70、11.89、12.09、12.22、12.36、12.62、12.83、13.08、13.18、13.50、13.73、14.04、14.24、14.46 and 14.73 min,RSD<0.2%.The characteristic analysis showed that HMW-GSs in HPCE spectrum have a boundary phenomenon.The X-type subunit region covers 12.36 to14.76 min,the Y-type region covers 9.39 to 12.36 min,and subunit 1Bx17 is the boundary point.In addition,the migration scenario of partial subunits in Glu-1A loci are quite different from those in SDS-PAGE.The three criteria（migration sequence of subunits,standard migration time,and separation characteristics of HPCE spectrum）obtained from the experiment can be used for rapid identification of HMW-GS in wheat by HPCE.This research provided support for qualitative analysis of HMW-GS and germplasm screening of wheat.