小麦品质性状的生化遗传基础研究及LMW-GS基因的克隆与序列分析
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摘要
小麦加工品质改良已成为我国小麦育种的主要目标之一。特别是我国加入WTO以后，对小麦产品的质量提出了更高的要求，小麦品质改良的任务将更加艰巨和重要。因此，深入了解小麦加工品质性状形成的分子基础及遗传规律，为品质改良提供理论依据和科学指导，对加速我国小麦品质育种和优质小麦生产具有重要意义。本研究选用在麦谷蛋白5个基因位点(即Glu-B1、Glu-D1、Glu-A3、Glu-B3和Glu-D3位点)上均含不同等位基因的小麦品种Suneca和Cook杂交F_4代60个麦谷蛋白纯合系，及18个黄淮海优质小麦产业带国家小麦展示品种为试材，研究了麦谷蛋白Glu-1和Glu-3位点基因等位变异对籽粒聚合体蛋白粒度分布及面粉品质的影响、籽粒聚合体蛋白粒度分布与面粉品质特性的关系和小麦品质性状与籽粒一些生化性状间的关系；本研究还设计合成了小麦A、B和D基因组中LMW-GS基因特异引物，并利用特殊遗传材料对其进行了验证和筛选；在此基础上，通过PCR方法克降了与优质相关的LMW-GS基因及其启子。现将主要研究结果简述如下：
①用Suneca和Cook杂交F_4代60个麦谷蛋白纯合系研究基因等位变异对籽粒聚合体蛋白粒度大小相对分布(用不溶聚合体蛋白占总聚合体蛋白含量的百分数表示，即UPP％)的影响，结果表明Glu-B1、Glu-D1、Glu-A3和Glu-B3位点等位基因变异对籽粒聚合体蛋白粒度大小相对分布的效应存在显著差异，含Glu-Blu、Glu-D1d、Glu-A3b和Glu-B3b基因的株系分别比含其等位基因Glu-B1i、Glu-D1a、Glu-A3d和Clu-B3h的株系有较大的UPP％；Glu-1位点和Clu-3位点对籽粒聚合体蛋白大小相对分布的影响存在累加和互作效应。
②Glu-D1、Glu-A3和Glu-B3位点上等位基因变异对面团形成时间(即揉面曲线图峰值对应的和面时问，简写PTM)有显著或极显著影响，含Glu-D1d,Glu-A3b和Glu-B3b基因的株系分别比含Glu-D1a，Glu-A3d和Glu-B3h基因的株系有较长的和面时间；在该遗传背景下，麦谷蛋白各基因位点对PTM的效应大小依次排列为：Glu-D1
小麦品质性状的生化遗传基础研究及LMW一GS基因的克隆与序列分析
>侧ues方J>‘ju训3>臼u一了=‘lueeD口。Glu一了位点和侧u一3位点对和面特性的影响存
在累加效应和互作效应。小麦籽粒聚合体蛋白粒度相对分布(UPP%)与曲粉的和血时
I、11(I，‘I’M)号毛极显著正才f}关，与lfll-粉蛋I全I质含量(I了既)才f}比，UI’I)%对l}‘l、M的彭llll’4更大些，
可作为一育利，早代品质性状选择的一个指标。
③以黄淮麦区品质差异较大的18个小麦品种为材料研究小麦品.质性状与籽粒
一些生化性状间的关系，结果表明面粉总蛋白质含量、总聚合体蛋白含量和不溶性
大聚合体蛋白含量3个生化性状对籽粒硬度、沉降值和湿曲筋含量这3个，{占质指标
有极人正!;刁作用:影响湿面筋含量(Y.)的关键生化性状是面粉总蛋白含量(xl)，其
!日!归方程为Y一魂.3054X一18.5971，相关系数是0.9992，达极显著水平;影!响沉降值
(丫.)的关键生化性状是总聚合体蛋白含量(X:)和大聚合体占总蛋自含量的百分比
(X，)，其1叫少_!方程为丫，沼.278魂X尹0.4930凡一54.8929，相关系数是0.7」98，也达到
极显著水〕}气
④根据GeneBank，喇眺等库中公布的所有己知LMW一GS荃囚不同区域的保守序
夕J设计了5对小麦A、13和D丛因组‘!，IJMw一GS华因特异引物，j!川六倍体普通小麦阿
勃二体、IA、1B和lD缺体，四倍体小麦及二倍体的一粒小麦和节节麦等特殊遗传材
料对其进行了PCI之验证和筛选。结果表明引物3(序列为5’叮GT八GAAACTGCCATCC’I、T3
’)和引物魂(5’Gl’CACCGCI’GCA’I’CGACA‘I’A3’)为麦谷蛋自脚:，双了位点l」MW一GS墓
因特异引物，其}’CI丈扩增产物约1600bp，包括了启动子卜_和完整编码区;引物5(5
’I’CC‘l’(;A(;AA(;I’(;CAI’GACA‘l’(;3‘)不l一7(5‘〔;『l’A(;GCACCAAC‘l、CCGGI’GC3’)足麦谷蛋白
(，，/乙了一/]，了位点IMw一(;S么誓I引特异I’Cl受弓I物，工琴I’Cl之JJ”增产二物约l/15()l〕I〕，包括了J‘t}动r不11
整个编码区。
⑤用引物3和4，通过I〕CR技术克隆的小堰6号和队优225(，’j:，一以了位点LMw一GS
琴比】)补列(其Ge;lel3a。k登录一号分别为AY263:亏69和AY299通85)与已报道的LMW一GS丛
囚序列高度}司源;克隆〔l{J这2个从因均包括了完整编码区和其!:游的胚乳特异性表达
启动J气:陕优225中该基因读码框为6训二15181)p，i(lJ小似6号，1，该从囚读码框为
606二1 520bl，，推测蛋自均为3O4aa(含N一端的ZOaa信号)Jk，成熟蛋自仅为284oa)。
陕优2巧的推测蛋白氦墓酸序列中含有8个CyS残丛，与所有报道的LMW一CS相同;
而小f僵6号的则多含l个CyS残墓;这是迄今首次发现的编码含9个Cys残基的LMw一GS
华囚。根据小麦谷蛋自聚合体粒度分布与品质间的关系研究结果，该荃因应属于优质
从因。这可能是小惬6号加工况.质优良的主要原因之一。
这些研究结果将为小麦育种实践中品质性状的预测和筛选及利用垫因工程方法
改良小麦品质提供理论依据。
Quality improvement has become a major goal of wheat breeding program in our country. The research of molecular mechanism and genetics pattern of wheat quality traits can provide with scientific basis for breeding practice. In this study, a set of 60 lines homozygous at glutenin loci, Glu-1 and Glu-3 from the F4 progeny population of Suneca X Cook , and 18 wheat cultivars with very different quality traits from Huang-Huai Wheat Region were chosen to investigate individual and combined effects of alleles at these loci on wheat quality properties , study the relationship between the size distribution of glutenin polymeric protein and wheat flour mixing properties and the relationship between some biochemical characters and quality traits. More over, the primers specific for LMW-GS genes at Glu-D3and Glu-B3 complex loci in wheat were developed, the genes at Glu-D3 complex loci in bread wheat cultivars were cloned and characterized. The major results are as follows: CD The parental cultivars Suneca and Cook had contrasting alleles at each of the five glutenin subunits loci (Glu-B1, G1u-D1, Glu-A3, Glu-B3 and Glu-D3), thus, a set of 60 lines homozygous of these loci from the F4 progenies of Suneca Cook was chosen to study the effects of allelic variation at glutenin complex loci on the size distributions of polymeric protein (measured by SE-HPLC) of wheat grain. The results showed that glutenin allelic variation at Glu-Bl, Glu-Dl, Glu-A3, Glu-B3 complex loci significantly affect the relative size distributions of polymeric protein (i. e. percentage of unextractable polymeric protein in the total polymeric protein, or UPP%). Allele Glu-Blu, Glu-Dld Glu-A3b and Glu-B3b was associated with greater UPP% value than allele Glu-Bli, Glu-D1a, Glu-A3d and Glu-B3h respectively . However , alleles (e and b) at the Glu-D3 locus did not give significantly different UPP % values. The effect of alleles at Glu-l and Glu-3 locus on the relative size distributions of polymeric protein appears cumulative and
interactive.
(2) Allelic variation at Glu-D1, Glu-A3 and Glu-B3 glutenin loci had significant effects on flour mixing time, the progenies with allele d at the Glu-Dl locus had significantly longer PTM than allele a, similarly Glu-A3b and Glu-B3b allele, respectively gave greater PTM than Glu-A3d and Glu-B3h. Alleles at the other loci (Glu-Bl and Glu-D3) did not give significantly different PTM. In this genetic background, the effect of different glutenin subunit loci on PTM could be ranked as Glu-Dl>Glu-B3> Glu-A3> Glu-Bl=Glu-D3. The effect of Glu-1 and Glu-3 alleles appears cumulative and interactive. In additions, Flour mixograph shape was closely related to UPP% value, UPP% was very strongly positive correlation with PTM. Compared with flour protein content (FP%) , UPP% gave greater effect on PTM, i. e. flour mixing properties, and it can be considered as one of criteria for quality selecting from early generation of breeding program.
(3) 18 wheat cultivars with very different quality traits were chosen to investigate the relationship between some biochemical characters and quality traits. The results indicated that total flour protein, total polymeric protein and unextractable macropolymeric protein content contributed significantly and positively to quality traits, i.e. grain hardness , sedimentation value and wet gluten content. The double-stepwise regression analysis showed that total polymeric protein content (X2) and the percent of macropolymeric glutenin in total polymeric protein (X6) were the most important factors influenceing sedimentation value (Y3), its regression equation was Y3= 8. 2784X2+0.4930 X6-54. 8929, while flour protein content (X1) was the key factor determining wet gluten content (Y4), the regression equation was Y4= 4. 3054X1 -18. 5971. The correlation coefficient of these two regression equations was 0.7498 and 0. 9992, respectively, and both were at very significant level.
(4) Based on the LMW-GS gene sequences reported in GenBank, the primer 1-9 for specific genome locus LMW-GS genes wer
①用Suneca和Cook杂交F_4代60个麦谷蛋白纯合系研究基因等位变异对籽粒聚合体蛋白粒度大小相对分布(用不溶聚合体蛋白占总聚合体蛋白含量的百分数表示，即UPP％)的影响，结果表明Glu-B1、Glu-D1、Glu-A3和Glu-B3位点等位基因变异对籽粒聚合体蛋白粒度大小相对分布的效应存在显著差异，含Glu-Blu、Glu-D1d、Glu-A3b和Glu-B3b基因的株系分别比含其等位基因Glu-B1i、Glu-D1a、Glu-A3d和Clu-B3h的株系有较大的UPP％；Glu-1位点和Clu-3位点对籽粒聚合体蛋白大小相对分布的影响存在累加和互作效应。
②Glu-D1、Glu-A3和Glu-B3位点上等位基因变异对面团形成时间(即揉面曲线图峰值对应的和面时问，简写PTM)有显著或极显著影响，含Glu-D1d,Glu-A3b和Glu-B3b基因的株系分别比含Glu-D1a，Glu-A3d和Glu-B3h基因的株系有较长的和面时间；在该遗传背景下，麦谷蛋白各基因位点对PTM的效应大小依次排列为：Glu-D1
小麦品质性状的生化遗传基础研究及LMW一GS基因的克隆与序列分析
>侧ues方J>‘ju训3>臼u一了=‘lueeD口。Glu一了位点和侧u一3位点对和面特性的影响存
在累加效应和互作效应。小麦籽粒聚合体蛋白粒度相对分布(UPP%)与曲粉的和血时
I、11(I，‘I’M)号毛极显著正才f}关，与lfll-粉蛋I全I质含量(I了既)才f}比，UI’I)%对l}‘l、M的彭llll’4更大些，
可作为一育利，早代品质性状选择的一个指标。
③以黄淮麦区品质差异较大的18个小麦品种为材料研究小麦品.质性状与籽粒
一些生化性状间的关系，结果表明面粉总蛋白质含量、总聚合体蛋白含量和不溶性
大聚合体蛋白含量3个生化性状对籽粒硬度、沉降值和湿曲筋含量这3个，{占质指标
有极人正!;刁作用:影响湿面筋含量(Y.)的关键生化性状是面粉总蛋白含量(xl)，其
!日!归方程为Y一魂.3054X一18.5971，相关系数是0.9992，达极显著水平;影!响沉降值
(丫.)的关键生化性状是总聚合体蛋白含量(X:)和大聚合体占总蛋自含量的百分比
(X，)，其1叫少_!方程为丫，沼.278魂X尹0.4930凡一54.8929，相关系数是0.7」98，也达到
极显著水〕}气
④根据GeneBank，喇眺等库中公布的所有己知LMW一GS荃囚不同区域的保守序
夕J设计了5对小麦A、13和D丛因组‘!，IJMw一GS华因特异引物，j!川六倍体普通小麦阿
勃二体、IA、1B和lD缺体，四倍体小麦及二倍体的一粒小麦和节节麦等特殊遗传材
料对其进行了PCI之验证和筛选。结果表明引物3(序列为5’叮GT八GAAACTGCCATCC’I、T3
’)和引物魂(5’Gl’CACCGCI’GCA’I’CGACA‘I’A3’)为麦谷蛋自脚:，双了位点l」MW一GS墓
因特异引物，其}’CI丈扩增产物约1600bp，包括了启动子卜_和完整编码区;引物5(5
’I’CC‘l’(;A(;AA(;I’(;CAI’GACA‘l’(;3‘)不l一7(5‘〔;『l’A(;GCACCAAC‘l、CCGGI’GC3’)足麦谷蛋白
(，，/乙了一/]，了位点IMw一(;S么誓I引特异I’Cl受弓I物，工琴I’Cl之JJ”增产二物约l/15()l〕I〕，包括了J‘t}动r不11
整个编码区。
⑤用引物3和4，通过I〕CR技术克隆的小堰6号和队优225(，’j:，一以了位点LMw一GS
琴比】)补列(其Ge;lel3a。k登录一号分别为AY263:亏69和AY299通85)与已报道的LMW一GS丛
囚序列高度}司源;克隆〔l{J这2个从因均包括了完整编码区和其!:游的胚乳特异性表达
启动J气:陕优225中该基因读码框为6训二15181)p，i(lJ小似6号，1，该从囚读码框为
606二1 520bl，，推测蛋自均为3O4aa(含N一端的ZOaa信号)Jk，成熟蛋自仅为284oa)。
陕优2巧的推测蛋白氦墓酸序列中含有8个CyS残丛，与所有报道的LMW一CS相同;
而小f僵6号的则多含l个CyS残墓;这是迄今首次发现的编码含9个Cys残基的LMw一GS
华囚。根据小麦谷蛋自聚合体粒度分布与品质间的关系研究结果，该荃因应属于优质
从因。这可能是小惬6号加工况.质优良的主要原因之一。
这些研究结果将为小麦育种实践中品质性状的预测和筛选及利用垫因工程方法
改良小麦品质提供理论依据。
Quality improvement has become a major goal of wheat breeding program in our country. The research of molecular mechanism and genetics pattern of wheat quality traits can provide with scientific basis for breeding practice. In this study, a set of 60 lines homozygous at glutenin loci, Glu-1 and Glu-3 from the F4 progeny population of Suneca X Cook , and 18 wheat cultivars with very different quality traits from Huang-Huai Wheat Region were chosen to investigate individual and combined effects of alleles at these loci on wheat quality properties , study the relationship between the size distribution of glutenin polymeric protein and wheat flour mixing properties and the relationship between some biochemical characters and quality traits. More over, the primers specific for LMW-GS genes at Glu-D3and Glu-B3 complex loci in wheat were developed, the genes at Glu-D3 complex loci in bread wheat cultivars were cloned and characterized. The major results are as follows: CD The parental cultivars Suneca and Cook had contrasting alleles at each of the five glutenin subunits loci (Glu-B1, G1u-D1, Glu-A3, Glu-B3 and Glu-D3), thus, a set of 60 lines homozygous of these loci from the F4 progenies of Suneca Cook was chosen to study the effects of allelic variation at glutenin complex loci on the size distributions of polymeric protein (measured by SE-HPLC) of wheat grain. The results showed that glutenin allelic variation at Glu-Bl, Glu-Dl, Glu-A3, Glu-B3 complex loci significantly affect the relative size distributions of polymeric protein (i. e. percentage of unextractable polymeric protein in the total polymeric protein, or UPP%). Allele Glu-Blu, Glu-Dld Glu-A3b and Glu-B3b was associated with greater UPP% value than allele Glu-Bli, Glu-D1a, Glu-A3d and Glu-B3h respectively . However , alleles (e and b) at the Glu-D3 locus did not give significantly different UPP % values. The effect of alleles at Glu-l and Glu-3 locus on the relative size distributions of polymeric protein appears cumulative and
interactive.
(2) Allelic variation at Glu-D1, Glu-A3 and Glu-B3 glutenin loci had significant effects on flour mixing time, the progenies with allele d at the Glu-Dl locus had significantly longer PTM than allele a, similarly Glu-A3b and Glu-B3b allele, respectively gave greater PTM than Glu-A3d and Glu-B3h. Alleles at the other loci (Glu-Bl and Glu-D3) did not give significantly different PTM. In this genetic background, the effect of different glutenin subunit loci on PTM could be ranked as Glu-Dl>Glu-B3> Glu-A3> Glu-Bl=Glu-D3. The effect of Glu-1 and Glu-3 alleles appears cumulative and interactive. In additions, Flour mixograph shape was closely related to UPP% value, UPP% was very strongly positive correlation with PTM. Compared with flour protein content (FP%) , UPP% gave greater effect on PTM, i. e. flour mixing properties, and it can be considered as one of criteria for quality selecting from early generation of breeding program.
(3) 18 wheat cultivars with very different quality traits were chosen to investigate the relationship between some biochemical characters and quality traits. The results indicated that total flour protein, total polymeric protein and unextractable macropolymeric protein content contributed significantly and positively to quality traits, i.e. grain hardness , sedimentation value and wet gluten content. The double-stepwise regression analysis showed that total polymeric protein content (X2) and the percent of macropolymeric glutenin in total polymeric protein (X6) were the most important factors influenceing sedimentation value (Y3), its regression equation was Y3= 8. 2784X2+0.4930 X6-54. 8929, while flour protein content (X1) was the key factor determining wet gluten content (Y4), the regression equation was Y4= 4. 3054X1 -18. 5971. The correlation coefficient of these two regression equations was 0.7498 and 0. 9992, respectively, and both were at very significant level.
(4) Based on the LMW-GS gene sequences reported in GenBank, the primer 1-9 for specific genome locus LMW-GS genes wer
引文
1 徐静斐，孙五成，程融 等编著．数量遗传学与水稻育种．安徽科技出版社，1990：PP43-46．
2 Gupta R B , etal.J. Cereal Sci, 1991, 14: 105-109．
3 Singh N K, Donovan C R, MacRitchie F. Use of sonication and size-exclusion high-performance liquid chromatography in the study of wheat flour proteins relative quantity of glutenin as a measure of bread-making quality. Cereal Chemistry. 1990,
(67) : 161-170．
4 Gupta R B, Khan K, MacRitchie F. Biochemical basis of flour properties in bread wheats. Effects of variation in the quantityand size distribution of polymeric protein. Joumal of Cereal Sci. 1993, (18) : 23-41．
5 Shepherd K W. In. Proc. 7th International Wheat Genetics Symposium' (T E Miller and R M D Koebner eds). Cambridge, England, 1988,PP919-931．
6 MacRitchie F, D L DuCros, C W Wrigley. Flour polypeptides related to wheat quality. In. Y Pomeranz (ed.). Advances in Cereal Science and Technology. 1990, 10:709-786．
7 Comec M, Y Popineau, J Lefebvre. Characterisation of gluten subfractions by SE-HPLC and dynamic Theological analysis in shear. J. Cereal Science. 1994, 19, 131-139．
8 Gupta, R B, K W Shepherd. Two-step one-dimentional SDS-PAGE analysis of LMW subunits of glutenin. Variation and genetic control of the subunits in hexploid wheat. Theor. Appl. Genet. 1990, 80:65-74．
9 Payne P I, et al. Catalogue of alleles for the complex gene loci, Glu-A1, Glu-B1, Glu-D1, which code for the high-molecular-weight subunits in hexploid wheat. Cereal Res. Communications. 1983,11 : 29-35．
10 Gupta R B, MacRitchie F. Allelic variation at glutenin subunit and gliadin loci, Glu-l,Glu-3 and Gli-1 of common wheats. Biochemical basis of the allelic effects on dough properties. J. of Cereal Sci. 1994, (19) :19-29．
11 Moonen J E, Scheepstra A, Graveland A. Biochemical properties of some high molecular weight subunits of wheat glutenin. J. Cereal Sci. 1985,3:17-27．
12 Batey L L, Gupta R B, MacRitchie F. Use of size-exclusion high-performance liquid chromatography in the study of wheat flour proteins: An improved chromatographic procedure. Cereal Chemistry. 1991, (68) :207-209．
13 Gupta R B, Paul J G, Cornish G B, et al. Allelic variation at glutenin subunit and gliadin loci, Glu-1 , Glu-3 and Gli-1 of common wheat. Its additive and interaction effects on dough properties. J. Cereal Sci. 1994, (19) :9-17．
14 Sing NK, et al. J. Cereal Sci, 1991, 14: 203-208．
2 Gupta R B , etal.J. Cereal Sci, 1991, 14: 105-109．
3 Singh N K, Donovan C R, MacRitchie F. Use of sonication and size-exclusion high-performance liquid chromatography in the study of wheat flour proteins relative quantity of glutenin as a measure of bread-making quality. Cereal Chemistry. 1990,
(67) : 161-170．
4 Gupta R B, Khan K, MacRitchie F. Biochemical basis of flour properties in bread wheats. Effects of variation in the quantityand size distribution of polymeric protein. Joumal of Cereal Sci. 1993, (18) : 23-41．
5 Shepherd K W. In. Proc. 7th International Wheat Genetics Symposium' (T E Miller and R M D Koebner eds). Cambridge, England, 1988,PP919-931．
6 MacRitchie F, D L DuCros, C W Wrigley. Flour polypeptides related to wheat quality. In. Y Pomeranz (ed.). Advances in Cereal Science and Technology. 1990, 10:709-786．
7 Comec M, Y Popineau, J Lefebvre. Characterisation of gluten subfractions by SE-HPLC and dynamic Theological analysis in shear. J. Cereal Science. 1994, 19, 131-139．
8 Gupta, R B, K W Shepherd. Two-step one-dimentional SDS-PAGE analysis of LMW subunits of glutenin. Variation and genetic control of the subunits in hexploid wheat. Theor. Appl. Genet. 1990, 80:65-74．
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