野生二粒小麦高分子量谷蛋白亚基等位变异分析
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摘要
小麦胚乳贮藏蛋白成分对小麦的品质具有重要作用,尤其是占贮减蛋白主要组分的小麦谷蛋白。小麦谷蛋白由高分子量谷蛋白亚基(HMW-GS)和低分子量谷蛋白亚基(LMW-GS)两部分组成;HMW-GS与LMW-GS的含量及组成可直接影响小麦面粉的加工和烘烤品质。本研究以由德国慕尼黑工业大学生命科学中心分子遗传室提供的175份野生二粒小麦为材料,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)方法和质谱技术对上述材料的HMW-GS组成进行鉴定和分析,以期鉴定和筛选一些新的谷蛋白亚基,为进一步克隆优质蛋白基因奠定基础,为今后研究野生二粒小麦HMW-GS与小麦品质的关系提供研究基础,并为探索利用野生二粒小麦上的特异HMW-GS改良小麦品质的新途径提供理论依据。主要研究结果如下:
l HMW-GS的SDS-PAGE分离鉴定
利用单向一步SDS-PAGE技术分析了供试材料的高分子量麦谷蛋白亚基等位变异,在Glu-1位点共检测到36种变异类型,其中Glu-A1位点有4种类型:1、1~*、2~*和Null,出现频率分别为45.14%、2.86%、4.57%和47.43%;Glu-B1位点有32种类型,比普通小麦1B染色体上HMW-GS的变异类型丰富得多,其中7+8和14.1+15.1亚基分布频率最高(20.00%),且有7个新亚基:1.1、5~*、7~*、12~*、14.1、15.1和16~*。供试小麦材料Glu-1位点的LMW-GS组合共有44种类型,以(null,14.1+15.1)组合为主要类型,占12.58%。该结果为进一步进行小麦亚基研究与品质改良提供了有意义的参考资料。
l HMW-GS的MALDI-TOF-MS鉴定
本研究对野生二粒小麦高分子量谷蛋白亚基采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)鉴定技术进行了优化,初步摸索出一套样品用量少、操作简便快速、适合高通量分析的技术体系。分析结果表明,15-20mg小麦种子样品足够进行质谱鉴定。图谱显示,不同高分子量蛋白组分分辨度较好,图谱清晰,分子量多集中在60-100kD之间。1Bx14.1、1By15.1、1Bx7~*、1By12~*和1Bx1.1这5个新亚基的质谱测定的分子量分别为82.9419kD、73.3575kD、86.1045kD、71.0637kD和88.4767kD。
Wheat seed storage proteins,especially glutenin,are important for processing-quality of wheat flour.The glutenins are the major components of wheat storage proteins,which consist of high-molecular-weight glutenin subunits(HMW-GS) and low-molecular-weight glutenin subunits(LMW-GS).It is well known that the composition and content of HMW and LMW glutenin subunits could influence the flour processing and bread-making quality.Genetic diversity of high molecular weight glutenin subunits(HMW-GS) encoded by the Glu-A1 and Glu-B1 loci were investigated by using one-dimensional sodium dodecylsulphate polyacrylamide gel electrophoresis(SDS-PAGE) and mass spectrometry(MS) in 175 wild emmer accessions which were from German Munich Industrial university.These datas may facilitate basic research on the relationship of industrial quality with HMW glutenin subunit and could be used to broaden the genetic basis for wheat breeding.The main results obtained were as the followings:
Separation and identification of HMW-GS by SDS-PAGE
HMW-GSs in Triticum dicoccoides were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) technique.36 allelic variations were found at Glu-1 locus,and four alleles were at the Glu-A1 locus:1,1~*,2~* and Null,32 alleles were at the Glu-B1 locus,a high degree of variation was evident for the HMW-GS in wild emmer accessions. The distribution of alleles appeared to be non random,the frequency of 7+8 and 14.1+15.1 were 20.0%,which were higher than others at the Glu-B1 locus.7 new allelic variations were found at Glu-B1 locus:1.1,5~*,7~*,12~*,14.1,15.1 and 16~*.Forty-four HMW glutenin subunit combinations have been observed at Glu-1 loci,of which the combinations(null,14.1+15.1) have accounted up to 12.58%.The results indicated that the combination of HMW is abundant among the tested wheat cultivars.
Identification of HMW-GS by MALDL-TOF-MS
The accurate measurement of molecular weights and characterization of wheat HMW-GS through matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was established in this study,which has the advantages of fast,simplified process,requirement of small samples and high resolution.The results show that HMW-GS components could be well characterized in the molecular weight range of 60-100kD.The molecular weight of the new subunits 1Bxl4.1,1By15.1,1Bx7~*,1By12~* and 1Bx1.1 which identified by MALDI-TOF-MS were 82.9419kD,73.3575kD,86.1045kD,71.0637kD and 88.4767kD respectively.
l HMW-GS的SDS-PAGE分离鉴定
利用单向一步SDS-PAGE技术分析了供试材料的高分子量麦谷蛋白亚基等位变异,在Glu-1位点共检测到36种变异类型,其中Glu-A1位点有4种类型:1、1~*、2~*和Null,出现频率分别为45.14%、2.86%、4.57%和47.43%;Glu-B1位点有32种类型,比普通小麦1B染色体上HMW-GS的变异类型丰富得多,其中7+8和14.1+15.1亚基分布频率最高(20.00%),且有7个新亚基:1.1、5~*、7~*、12~*、14.1、15.1和16~*。供试小麦材料Glu-1位点的LMW-GS组合共有44种类型,以(null,14.1+15.1)组合为主要类型,占12.58%。该结果为进一步进行小麦亚基研究与品质改良提供了有意义的参考资料。
l HMW-GS的MALDI-TOF-MS鉴定
本研究对野生二粒小麦高分子量谷蛋白亚基采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)鉴定技术进行了优化,初步摸索出一套样品用量少、操作简便快速、适合高通量分析的技术体系。分析结果表明,15-20mg小麦种子样品足够进行质谱鉴定。图谱显示,不同高分子量蛋白组分分辨度较好,图谱清晰,分子量多集中在60-100kD之间。1Bx14.1、1By15.1、1Bx7~*、1By12~*和1Bx1.1这5个新亚基的质谱测定的分子量分别为82.9419kD、73.3575kD、86.1045kD、71.0637kD和88.4767kD。
Wheat seed storage proteins,especially glutenin,are important for processing-quality of wheat flour.The glutenins are the major components of wheat storage proteins,which consist of high-molecular-weight glutenin subunits(HMW-GS) and low-molecular-weight glutenin subunits(LMW-GS).It is well known that the composition and content of HMW and LMW glutenin subunits could influence the flour processing and bread-making quality.Genetic diversity of high molecular weight glutenin subunits(HMW-GS) encoded by the Glu-A1 and Glu-B1 loci were investigated by using one-dimensional sodium dodecylsulphate polyacrylamide gel electrophoresis(SDS-PAGE) and mass spectrometry(MS) in 175 wild emmer accessions which were from German Munich Industrial university.These datas may facilitate basic research on the relationship of industrial quality with HMW glutenin subunit and could be used to broaden the genetic basis for wheat breeding.The main results obtained were as the followings:
Separation and identification of HMW-GS by SDS-PAGE
HMW-GSs in Triticum dicoccoides were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) technique.36 allelic variations were found at Glu-1 locus,and four alleles were at the Glu-A1 locus:1,1~*,2~* and Null,32 alleles were at the Glu-B1 locus,a high degree of variation was evident for the HMW-GS in wild emmer accessions. The distribution of alleles appeared to be non random,the frequency of 7+8 and 14.1+15.1 were 20.0%,which were higher than others at the Glu-B1 locus.7 new allelic variations were found at Glu-B1 locus:1.1,5~*,7~*,12~*,14.1,15.1 and 16~*.Forty-four HMW glutenin subunit combinations have been observed at Glu-1 loci,of which the combinations(null,14.1+15.1) have accounted up to 12.58%.The results indicated that the combination of HMW is abundant among the tested wheat cultivars.
Identification of HMW-GS by MALDL-TOF-MS
The accurate measurement of molecular weights and characterization of wheat HMW-GS through matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) was established in this study,which has the advantages of fast,simplified process,requirement of small samples and high resolution.The results show that HMW-GS components could be well characterized in the molecular weight range of 60-100kD.The molecular weight of the new subunits 1Bxl4.1,1By15.1,1Bx7~*,1By12~* and 1Bx1.1 which identified by MALDI-TOF-MS were 82.9419kD,73.3575kD,86.1045kD,71.0637kD and 88.4767kD respectively.
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