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分离小麦麦谷蛋白亚基的几个关键因素研究 预览

Several Key Factors for Isolating the Glutenin Subunits of Wheat
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摘要 小麦高分子量、低分子量麦谷蛋白亚基组成是影响小麦加工品质的重要因素,为了经济快速鉴定小麦高低分子量麦谷蛋白亚基组成,需要不断对提取和分离这些蛋白的方法进行优化。在Singh等提出的提取分离小麦高低分子量麦谷蛋白亚基方法的基础上,对其中涉及的单体蛋白去除、麦谷蛋白还原和烷化过程中使用的异丙醇浓度、烷化剂浓度以及烷化过程能否简化几个关键因素进行了深入研究。结果表明:在麦谷蛋白还原时,在10%~50%的异丙醇浓度范围内,不同浓度的异丙醇对高分子量麦谷蛋白亚基的提取效果没有差别,而低分子量麦谷蛋白亚基在用低浓度异丙醇提取时效果较差,随异丙醇浓度提高,提取效果逐渐提高,异丙醇浓度提高到30%时,提取效果达到最高,异丙醇浓度继续提高到50%,提取效果不再提高;使用30%和50%的异丙醇,去除单体蛋白的效果相同;把烷化剂直接加到样品缓冲液中进行烷化,不同浓度(0.6%~1.4%)的烷化剂处理麦谷蛋白亚基的烷化效果相同,但烷化剂浓度为1.4%时电泳背景较重。优化后的方法为:在单体蛋白去除和麦谷蛋白还原时把异丙醇浓度由原来的50%降为30%,去掉单独的烷化步骤,把0.6%的烷化剂直接加到样品缓冲液中进行烷化。优化后的方法不但减少了试剂用量,简化了提取步骤,还提高了电泳条带强度。 The composition of wheat high and low molecular weight glutenin subunits is an important factor affecting the processing quality of wheat.In order to quickly and economically identify the composition of wheat high and low molecular weight glutenin subunits,it is necessary to continuously optimize the methods of extracting and isolating these proteins.Based on the method of extracting and separating wheat high and low molecular weight glutenin subunits proposed by Singh et al.,we conducted in-depth research on several key factors like isopropyl alcohol concentration,alkylating agent concentration and extraction steps used in monomeric protein removal,gluten reduction and alkylation.The results showed that there were no difference in the extraction effect of high molecular weight glutenin subunits between different concentrations of isopropanol treatments in the range of 10%-50%during the reduction of glutenin.When the low molecular weight glutenin subunit was extracted with low concentration of isopropanol,the effect was poor.With the increase of isopropanol concentration,the extraction effect gradually increased.When the concentration of isopropanol was increased to 30%,the extraction effect reached the highest,but when the concentration of isopropanol continued to increase to 50%,the effect was no longer improved.Using 30%and 50%isopropanol,the effect of removing the monomeric proteins was the same.When the alkylating agent was directly added to the sample buffer for alkylation,different concentrations(0.6%-1.4%)of alkane treatments had the same alkylation effects for the glutenin subunits,but when the concentration of alkylating agent was 1.4%,the electrophoresis background was heavier.The optimized method was to reduce the isopropanol concentration from 50%to 30%during monomer protein removal and gluten reduction,remove the separate alkylation step,and add 0.6%alkylating agent directly to the sample buffer for alkylation.The optimized method not only reduced the amount of reagents,but also simplified the extra
作者 李春燕 张翠绵 柴建芳 秘彩莉 马秀英 赵和 吕孟雨 周硕 董福双 刘永伟 杨帆 王海波 LI Chunyan;ZHANG Cuimian;CHAI Jianfang;BI Caili;MA Xiuying;ZHAO He;LU Mengyu;ZHOU Shuo;DONG Fushuang;LIU Yongwei;YANG Fan;WANG Haibo(Institute of Genetics and Physiology,Hebei Academy of Agriculture and Forestry Sciences,Plant Genetic Engineering Center of Hebei Province,Shijiazhuang050051,China;College of Life Sciences,Hebei Normal University,Shijiazhuang050024,China)
出处 《华北农学报》 CSCD 北大核心 2019年第4期32-36,共5页 Acta Agriculturae Boreali-Sinica
基金 国家转基因生物新品种培育重大专项(2016ZX08002005) 河北省自然科学基金项目(C2014301005) 河北省现代农业科技创新工程(2019-4-8-1).
关键词 小麦 麦谷蛋白亚基 SDS-PAGE 优化 分离方法 Wheat Glutenin subunit SDS-PAGE Optimization Isolating method
作者简介 李春燕(1990-),女,河北临漳人,在读硕士,主要从事小麦分子育种研究;通讯作者:柴建芳(1964-),男,河北平乡人,研究员,博士,主要从事小麦分子育种研究;通讯作者:王海波(1958-),男,河北满城人,研究员,博士,博士生导师,主要从事作物生物技术研究。
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