簇毛麦新型HMW-GS的序列分析及加工品质效应鉴定

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杨华, 高翔, 陈其皎, 赵万春, 董剑, 李晓燕. 簇毛麦新型HMW-GS的序列分析及加工品质效应鉴定. 作物学报, 40(4): 600-610
YANG Hua, GAO Xiang, CHEN Qi-Jiao, ZHAO Wan-Chun, DONG Jian, LI Xiao-Yan. Isolation, Characterization and Farinograph Analysis of Novel HMW-GSs fromDasypyrum villosum . 作物学报, 40(4): 600-610 复制到剪切板

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作物学报编辑部

簇毛麦新型HMW-GS的序列分析及加工品质效应鉴定

杨华¹, 高翔^1,^2,^*, 陈其皎¹, 赵万春^1,^*, 董剑^1,², 李晓燕¹

¹西北农林科技大学农学院, 陕西杨凌 712100

²陕西省小麦工程技术研究中心 / 陕西省小麦新品种培育研究中心, 陕西杨凌 712100

^*通讯作者(Corresponding authors): 高翔, E-mail:gx@nwsuaf.edu.cn; 赵万春, E-mail:zhaowc2009@hotmail.com

收稿日期:2014-01-12

基金:本研究由“十二五”农村领域国家科技计划课题(2011AA100501), 国家现代农业产业技术体系建设专项(CARS-3-2-47), 国家自然科学基金项目(31171538, 30900896)和西北农林科技大学唐仲英育种基金项目(A212020912)资助。

摘要

利用设计的HMW-GS特异引物, 从簇毛麦TA10220中克隆到6条HMW-GS基因序列(1.0~1.7 kb, GenBank登录号为KF887414~KF887419), 比普通小麦HMW-GS基因序列小, 其中KF887414~KF887417为能正常表达的基因, KF887418和KF887419是编码区出现终止信号的假基因。综合推导氨基酸的序列分析以及基于编码蛋白全序列、N-端和C-端域的进化树分析, 认为KF887414属于y型HMW-GS, KF887415~KF887419的氨基酸序列同时具有x和y型的结构特征。将具有完整编码区的4个基因在宿主菌Escherichia coli Rosetta-gami B(DE3)中经IPTG诱导表达, 通过SDS-PAGE分析表达的蛋白质和种子提取的HMW-GS, 经Western blot进一步检测表达产物, 证明蛋白质表达成功。通过His-Trap HP柱纯化表达蛋白, 回收产物经SDS-PAGE分析后, 低温冷冻干燥备用。将冷冻干燥的回收产物加入中国春基础面粉中, 利用微量掺粉试验通过测定粉质参数来鉴定簇毛麦来源的HMW-GS对面粉品质的影响, 结果表明, 簇毛麦来源的4个HMW-GS对于面粉加工品质具有显著的正向效应。

关键词: 簇毛麦; 高分子量谷蛋白亚基; 原核表达; 掺粉实验

Isolation, Characterization and Farinograph Analysis of Novel HMW-GSs fromDasypyrum villosum

YANG Hua¹, GAO Xiang^1,^2,^*, CHEN Qi-Jiao¹, ZHAO Wan-Chun^1,^*, DONG Jian^1,², LI Xiao-Yan¹

¹ College of Agronomy, Northwest A&F University, Yangling 712100, China

² Wheat Engineering Research Center of Shaanxi Province / New Varieties Cultivation of Wheat Engineering Research Center of Shaanxi Province, Yangling 712100, China

Fund:

Abstract

Dasypyrum villosum carrying many novel HMW-GS alleles is an important genetic resource for wheat protein improvement. In this study, we isolated six HMW-GS genes fromD. villosum TA10220 (1.0-1.7 kb, GenBank accession numbers: KF887414-KF887419), which were substantially smaller than those from common wheat, using a pair of specific primers. An in-frame stop codon was found in the coding sequences of KF887418 and KF887419 and thus these genes might be pseudogenes. The comprehensive analysis of deduced amino acid sequence, and phylogenetic and evolutionary analyses of full sequence, N- and C-terminal domains revealed that KF887414 was closely related to y-type HMW-GS, but KF887415-KF887419 had structural characteristics of both x- and y-types. DNA fragments of KF887414-KF887417 were subcloned into the pEASY-E2 expression vector and expressed inEscherichia coli Rosetta-gami B(DE3) cell under IPTG induction. The four genes were successfully expressed inE. coli system according to SDS-PAGE analysis (both the expressed protein and HMW-GS isolated from seed) and western-blotting assay. The fusion protein was purified and recovered by His-Trap affinity chromatography and low temperature cryodesiccation, and then integrated into the control flour by using a 4 g Micro-dough LAB Farinograph. Results showed that the four HMW-GSs originated fromD. villosum had positive effects on dough quality property.

Keyword: Dasypyrum villosum; High-molecular-weight glutenin subunits (HMW-GS); Prokaryotic expression; Farinograph

Show Figures

小麦高分子量谷蛋白亚基(HMW-GS)和低分子量谷蛋白亚基(LMW-GS)通过分子内和分子间二硫键形成聚合蛋白, 从而影响面粉的加工品质^{[ 1]}。HMW-GS影响面团的弹性和面包的加工品质^{[ 2, 3]}。编码HMW-GS (60~120 kD)的基因位于第1部分同源群染色体1A、1B和1D长臂上^{[ 4, 5, 6, 7, 8, 9]}, 每个位点由2个紧密连锁的基因组成, 其中较大的基因编码x型HMW-GS的合成, 较小的基因编码y型HMW-GS的合成^{[ 9, 10]}。HMW-GS具有高度保守的5’和3’端序列, 编码区内部不存在内含子^{[ 11, 12]}。HMW-GS典型的结构由4部分组成, 分别是信号肽(蛋白质加工过程被删除)、高度保守的N-端和C-端以及中间重复区^{[ 2, 3]}。x和y型HMW-GS主要区别在于半胱氨酸残基的个数和位置, 中间重复区重复单元的类型, 其中六肽以PGQGQQ、九肽以GYYPTS/PLQQ的保守形式出现在所有x、y型亚基中, 而三肽以GQQ的保守形式只存在在x型亚基中^{[ 2, 3]}; 多数的半胱氨酸残基存在于N-端保守区, y型的有5个, 并且第3和第4个相邻, x型的有3个; C-端都只有1个; 但是1By9、1Dy10和1Dy12近C-端的中间重复区多了1个半胱氨酸残基, 而1Dx5近N-端的中间重复区多了1个半胱氨酸残基^{[ 3]}。

簇毛麦( Dasypyrum villosum, 2 n = 14, VV)属于禾本科小麦亚族簇毛麦属, 为一年生二倍体异花授粉草本植物, 具有抗多种病害、品质优良、抗旱耐寒、密穗、多花及分蘖能力强等特性, 还带有提高籽粒蛋白质含量、赖氨酸含量和面筋强度的基因, 是普通小麦遗传改良优异的三级基因源^{[ 13, 14]}。簇毛麦1V染色体对小麦加工品质具有正效应, 可以显著提高面筋的强度, 是位于1V染色体上 Glu-V1、 Gli-V1和 Glu-V13位点的作用^{[ 15, 16, 17, 18]}。其中1V染色体上有: 编码HMW-GS的 Glu-V1位点, 编码贫硫(ω型)和富硫(γ型)醇溶蛋白基因 Gli-V1位点和编码低分子谷蛋白 Glu-V3位点^{[ 15, 16, 17, 18, 19, 20]}。

利用同源克隆技术, 一些研究者从簇毛麦中分离到HMW-GS编码基因及其启动子序列, 并发现了没有功能的假基因^{[ 20, 21, 22, 23, 24]}。迄今, 尚缺乏对该基因家族成员多样性、序列特征、以及加工品质特征方面的深入报道。对簇毛麦HMW-GS基因序列和推导的氨基酸序列分析, 可为进一步了解簇毛麦HMW-GS与普通小麦HMW-GS的关系提供依据, 而且原核体外表达和功能鉴定有助于验证簇毛麦HMW-GS对小麦籽粒品质和加工品质的影响。

1 材料与方法

1.1 HMW-GS提取及SDS-PAGE分析

参考段淑娥等^{[ 25]}和Verbruggen等^{[ 26]}的方法, 从单粒种子中提取簇毛麦TA10220 (美国堪萨斯州大学小麦遗传资源研究中心提供) HMW-GS, 然后进行SDS-PAGE分析^{[ 25, 26]}。

1.2 目的基因的克隆与序列分析

以室内培养2周的簇毛麦TA10220幼嫩叶片为材料, 采用微量CTAB法提取基因组DNA^{[ 27]}。

根据GenBank公布的HMW-GS基因编码区的保守序列设计1对简并引物(hmw-F: 5'-TCATCACC CACAACACCGA-3'; hmw-R: 5'-TCACTGGCTRGC CGACAAT-3'), 以簇毛麦TA10220的基因组DNA为模板扩增目的基因。反应体系25 μL, 包含5× PrimeSTAR buffer (Mg²⁺ plus) 5 μL, dNTP Mixture (各2.5 mmol L^-1) 2 μL, 引物各1 μL, DNA模板1 μL, PrimeSTAR HS DNA Polymerase (2.5 U μL^-1, TaKaRa) 0.2 μL。PCR反应条件为94℃ 2 min; 98℃ 10 s, 68℃ 30 s, 72℃ 2 min, 共30个循环; 最后72℃延伸10 min。

PCR产物经琼脂糖凝胶电泳检测, 利用DNA凝胶回收试剂盒(天根生化科技北京有限公司)回收目的条带, 对回收的产物先进行末端加A反应, 其反应体系10 μL, 含回收产物4 μL, 2 mmol L^-1dATP 0.8 μL, 10×PCR buffer 1 μL, Taq DNA polymerase 0.3 μL; 然后与克隆载体pEASY-T1 (北京全式金生物技术有限公司)连接, 转入克隆菌株 E. coli Trans1-T1 (北京全式金生物技术有限公司)中, 使用T7/SP6通用引物对白色菌落进行PCR扩增, 筛选阳性克隆, 由南京金斯瑞生物科技有限公司测序。使用DNAMAN、MEGA5.0及NCBI和SignalP 4.1 Server网站上的在线工具对所得序列进行核苷酸及推导氨基酸序列分析。

1.3 目的基因的诱导表达与鉴定

根据测序结果设计2对表达引物(hmwExF1: 5'- GAAGGTGGGGCCTCTGC-3', hmwExR1: 5'-CTGG CTGACCGACAATGC-3'; hmwExF2: 5'-GAAGGCG AGGGCTCTGGG-3', hmwExR2: 5'-CTGGCTGGCC GACAATGC-3')。将表达引物扩增的目的基因与表达载体pEASY-E2 (北京全式金生物技术有限公司)连接, 转入克隆菌株 E. coli T rans1-T1中, 在LB固体培养基(含50 mg L^-1 AMP)上筛选阳性克隆。用T7F/hmwExR或hmwExF/T7R引物筛选具有正确表达方向的重组子, 经测序确定序列的正确性。提取质粒, 转入表达菌株 E. coli Rosetta-gami B(DE3)(北京全式金生物技术有限公司)中。挑取单个阳性克隆至LB液体培养基(含50 mg L^-1 AMP)中, 37℃过夜, 将培养物按1∶100接种至新鲜LB液体培养基(含50 mg L^-1 AMP)中, 37℃, 200转 min^-1培养至菌液OD₆₀₀值为0.5; 取1 mL菌液作为阴性对照, 向剩余菌液中加入IPTG (1 mmol L^-1), 37℃诱导6 h, 12 000 × g离心10 min收集菌体, 用SDS-PAGE (12%分离胶, 4%浓缩胶)分析蛋白表达结果。

1.4 表达产物的Western blot分析

处理后的蛋白样品经12% SDS-PAGE, 马上电转移至硝酸纤维素(NC)膜, 用新鲜配制的封闭液(5%脱脂奶粉), 在室温摇床封闭1 h, 弃封闭液; 以鼠抗His抗体为一抗(1∶10 000), 室温孵育1 h, 用TBS和TBST各洗膜2次, 每次15 min; HRP标记的羊抗鼠IgG为二抗(1∶10 000), 用TBS和TBST各洗膜2次, 每次15 min, 最后用DAB显色试剂盒进行化学发光显色。

1.5 目的蛋白的分离纯化

收集诱导表达菌液, 于4℃ 12 000 × g离心 10 min, 弃上清液, 加入10 mL破碎缓冲液(pH 8.5, 50 mmol L^-1 Tris-HCl, 2 mmol L^-1EDTA, 100 mmol L^-1 NaCl, 1 mg L^-1溶菌酶)重悬菌体。冰浴45 min后超声破碎, 离心收集沉淀。沉淀经过预处理后过柱纯化, 平衡缓冲液为50 mmol L^-1磷酸缓冲液(pH 7.4, 0.5 mol L^-1 NaCl, 20 mmol L^-1咪唑), 将洗脱缓冲液为50 mmol^-1磷酸缓冲液(pH 7.4, 0.5 mol L^-1 NaCl, 4 mol L^-1脲, 500 mmol L^-1咪唑), 收集的溶液透析24 h, 低温冷冻干燥保存待用。用SDS-PAGE (12%分离胶, 4%浓缩胶)分析蛋白纯化结果。

1.6 目的蛋白的功能鉴定

用4 g全自动微型粉质仪(Micro-dough LAB, Perten公司, 瑞典)测定粉质参数。参考国家标准《小麦吸水量和面团揉和性能测定法——粉质仪法(GB/T14614-93)》和HMW-GS功能的体外鉴定法, 设定如下程序。称基础面粉4 g, 与10 mg目的蛋白混合, 加入适量水和50 μg mL^-1 DTT 0.25 mL, 揉和0.5 min后加入200 μg mL^-1 KIO₃ 0.25 mL, 反应 4 min, 记录粉质仪曲线。参考Xu等^{[ 28]}和Chen等^{[ 29]}的描述分析粉质参数。

2 结果与分析

2.1 核苷酸序列分析

利用引物对hmw-F/hmw-R扩增簇毛麦TA10220基因组DNA, 获得1~2 kb的目标条带(图1)。回收该条带, 末端加A, 连接至pEASY-T1载体上, 转化到大肠杆菌Trans1-T1中, 经过菌落PCR扩增, 筛选出43个阳性克隆, 测序后通过序列比对, 得到6个非重复序列。用NCBI在线工具BLASTn对测序结果进行同源性分析, 表明测得6条序列与数据库中已经公布的一年生簇毛麦( D. villosum)、多年生簇毛麦( D. breviaristatum)、小麦及其亲缘植物的HMW-GS相似性很高。因此, 初步确定这6个序列为簇毛麦HMW-GS基因, 将序列提交GenBank (http://www.ncbi.nlm.nih.gov/sites/entrez), 登录号为KF887414至KF887419。

	Figure Option View Download New Window
	图1 2%琼脂糖凝胶电泳分析PCR产物M: 分子量标准DM2000; Negative: 无DNA模板的阴性对照; 箭头示目的带。Fig. 1 Amplified product separated on 2% agarose gelM: DNA marker DM2000; Negative: template DNA free; the arrows show target bands.

2.2 推导的氨基酸序列分析

用NCBI在线工具ORF Finder分析克隆获得的6条目标序列, KF887414、KF887415、KF887416和KF887417具有完整的开放阅读框(表1), 编码区内无内含子; KF887418和KF887419同样具有完整的开放阅读框, 编码区内也没有内含子的存在, 但在KF887418编码氨基酸的第323位和KF887419编码氨基酸的第269位分别出现提前终止信号(图2)。经多次测序鉴定表明, 这2个基因可能是假基因。

6条推导氨基酸序列均包括信号肽、N-端保守区、中间重复区和C-端保守区等HMW-GS典型的氨基酸序列结构特征(图2); 与小麦属A、B和D、山羊草属C和U、偃麦草属E、黑麦R和类大麦属K染色体组编码的HMW-GS氨基酸组成及结构特征相似(表2)。其中, 所有推测的氨基酸序列的信号肽均由21个氨基酸残基组成, 但在第8、第13、第16和第18氨基酸处分别出现C/L、V、L和F的多态性位点, 但SignalP分析表明, 这些多肽性位点不影

表1 核苷酸序列和氨基酸序列特征 Table 1 Basic characteristics of nucleotide sequence and amino acid sequence

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	图2 KF887414、KF887415、KF887416、KF887417、KF887418和KF887419推导氨基酸序列分析Fig. 2 Deduced amino acid sequences of KF887414, KF887415, KF887416, KF887417, KF887418, and KF887419

表2 不同来源的HMW-GS的主要结构特征的比较 Table 2 A summary of properties of the primary structure of HMW-GS from Dasypyrum villosumin comparison with some HMW-GS from common wheat and wheat-related grass

亚基 Subunit	来源 Origin	登录号 Accession number	编码区长度 Size of coding sequence (bp)	信号肽 Signal peptide Size (aa)	N-端保守区N-terminal region		中间重复区 Repetitive region		C-端保守区C-terminal region		总数 Total
亚基 Subunit	来源 Origin	登录号 Accession number	编码区长度 Size of coding sequence (bp)	信号肽 Signal peptide Size (aa)	长度 Size (aa)	Cys	长度 Size (aa)	Cys	长度 Size (aa)	Cys	长度 Size (aa)	Cys
1Ax1	T. aestivum	X61009	2490	21	86	3	681	0	42	1	830	4
1Bx7	T. aestivum	X13927	2367	21	81	3	645	0	42	1	789	4
1Bx14	T. aestivum	AY367771	2385	21	86	1	646	0	42	1	795	2
1Cx	Ae. markgrafii	AF476959	2385	21	86	3	646	0	42	1	795	4
1Dx2	T. aestivum	X03346	2514	21	88	3	687	0	42	1	838	4
1Dx5	T. aestivum	X12928	2517	21	89	3	687	1	42	1	839	5
1Ex2	T. elongatum	EF204546	2049	21	89	3	531	0	42	1	683	4
1Kx	C. delileana	AY804128	2046	21	86	3	533	0	42	1	682	4
1Ux	Ae. umbellulata	AF476961	2976	21	86	3	843	0	42	1	992	4
1Rx	S. cereale	AJ314782	2343	21	86	3	632	0	42	1	781	4
1Ay	T. aestivum	X03042	1803	21	104	5	455	0	42	1	601	6
1By9	T. aestivum	X61026	2115	21	104	5	538	1	42	1	705	7
1Cy	Ae. markgrafii	AF476960	1899	21	104	5	466	1	42	1	633	7
1Dy10	T. aestivum	X12929	1944	21	104	5	481	1	42	1	648	7
1Dy12	T. aestivum	BK006459	1974	21	104	5	491	1	42	1	658	7
1Ey	T. elongatum	AY280615	1524	21	105	5	340	1	42	1	508	7
1Ey2	T. elongatum	EF204547	1236	21	105	5	244	1	42	1	412	7
1Ky	C. delileana	AY834230	1725	21	105	5	407	1	42	1	575	7
1Uy	Ae. umbellulata	AF476962	1965	21	104	5	488	1	42	1	655	7
1Ry	S. cereale	AJ314767	2121	21	104	5	540	1	42	2	707	8
1V	D. villosum	FJ600492	1494	21	105	5	330	0	42	1	498	6
1V	D. villosum	AY608740	1467	21	102	4	329	0	37	1	489	5
1V	D. villosum	KF887414	1602	21	102	5	369	1	42	1	534	7
1V	D. villosum	KF887415	1233	21	105	5	243	0	42	1	411	6
1V	D. villosum	KF887416	1089	21	102	5	198	0	42	1	363	6
1V	D. villosum	KF887417	1089	21	102	5	198	0	42	1	363	6
1V	D. villosum	KF887418	1476	21	102	5	327	0	42	1	492	6
1V	D. villosum	KF887419	1314	21	105	5	270	1	42	1	438	7
AgeloG6	A. elongatum	AY263345	1425	21	104	5	308	0	42	1	475	6
AgeloG9	A. elongatum	AY264065	1488	21	105	5	328	1	42	1	496	7
AgeloG10	A. elongatum	DQ078274	1149	21	105	5	215	1	42	1	383	7

响其疏水性; 在N-端保守区, KF887415和KF887419由105个氨基酸残基组成, 与其相比KF887414、KF887416、KF887417和KF887418的N-端保守区由102个氨基酸残基组成, 缺少KGG这3个氨基酸残基, 表明这6条序列的N-端保守区具有y型HMW- GS的特征; 在中间重复区, 六肽和九肽等高度保守的结构单元重复出现, 但在KF887415、KF887416、KF887417、KF887418和KF887419的中间重复区多次出现类三肽PGQ, 具有x型HMW-GS的中间重复区特征, 而KF887414仅具有y型HMW-GS中间重复区的结构特征; 所推测的6条序列的C-端保守区均由42个氨基酸残基组成; KF887415、KF887416、KF887417和KF887418成熟的氨基酸序列(无信号肽)具有6个半胱氨酸残基, 而KF887414和KF887419有7个半胱氨酸残基, 其中, N-端保守区均有5个半胱氨酸残基, 第3和第4个相邻, 符合y型HMW- GS氨基酸序列N-端保守区的特点, C-端保守区均有1个半胱氨酸残基, 但在中间重复区, KF887414近C-端保守区的第420氨基酸处和KF887419的近N-端保守区的第167氨基酸处为半胱氨酸残基(图2和表2)。因此认为, KF887414属于y型HMW-GS, 而KF887415~KF887419为同时具有x和y型结构特征的混合亚基。

2.3 系统进化树分析

利用MEGA5.1软件, 对来自 D. villosum的6个克隆序列与来自小麦、山羊草属、偃麦草属、黑麦、类大麦属等不同基因组和已经公布的簇毛麦属V染色体组编码的HMW-GS氨基酸序列进行全序列、N-端保守区和C-端保守区聚类分析。在全序列聚类图中, 本试验获得的6个序列与y型HMW-GS的关系较近, 与x型HMW-GS的关系较远; 在y型HMW- GS类型中, 与小麦A、B和D、山羊草属的C和U、偃麦草属E、黑麦R和类大麦属K染色体组的亲缘关系较远, 但与簇毛麦属V染色体组的HMW-GS同属一支(图3-A)。N-端保守区聚类图显示与全序列分析结果相似(图3-B), 但C-端保守区分析结果略有不同: 除KF887414外的各序列与x型HMW-GS的C-端保守区的亲缘关系较近, 其中又与簇毛麦V的关系最近, 而与小麦A、B和D、山羊草属的C和U和黑麦R染色体组的亲缘关系较远, KF887414与y型的HMW-GS的关系较近, 尤其与小麦D和山羊草属U染色体组的亲缘关系较近(图3-C)。

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	图3 簇毛麦HMW-GS编码的蛋白序列系统演化分析(Neighbor-joining进化树)Fig. 3 Phylogenetic analysis of the deduced amino acid sequence of HMW-GS from Dasypyrum villosum (Neighbor-joining tree)

A: 全序列; B: N-端保守区; C: C-端保守区。

A: full length sequence; B: N-terminal region; C: C-terminal region.

2.4 表达产物的SDS-PAGE分析

由于KF887418和KF887419是序列内部有提前终止信号的假基因, 所以未对其进行原核表达。其他4个初步判定为有功能的基因, 根据目的序列的完整编码区, 设计2对表达引物, 筛选表达基因插入方向正确的阳性克隆并测序鉴定。然后加入IPTG诱导6 h, 表达载体和能够表达的基因的重组质粒在 E. coli Rosetta-gami B(DE3) 中表达, 表达载体上的His标签的大小为2.101 kD, 融合表达蛋白产物的分子质量如表1所示, 与电泳检测的蛋白质条带大小相似(图4-A); 原核表达的蛋白质与来源于种子的高分子谷蛋白亚基具有基本相同的迁移率(图4-B), 表明克隆的HMW-GS基因在大肠杆菌中正确表达。

2.5 目的基因表达产物的Western blot分析

重组质粒经诱导产生的蛋白质条带与His抗体产生特异杂交信号(图5), 表明目的蛋白基因得到了成功表达。

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图4 KF887414、KF887415、KF887416和KF887417基因的诱导表达产物和种子HMW-GS的SDS-PAGE分析M: 蛋白质分子质量标准Marker II。A图中, 1、3、5和7泳道为未诱导重组质粒表达产物; 2、4、6和8泳道为经IPTG诱导后的重组质粒的表达产物; 箭头示基因的表达产物。B图中, 泳道1为未诱导重组质粒表达产物; 2~5泳道为经IPTG诱导后的重组质粒的表达产物, 依次是 KF887414、KF887415、KF887416和KF887417基因; 泳道6为种子提取的HMW-GS; 箭头示目的蛋白。Fig. 4 Analyses of expressed proteins of genes from KF887414 to KF887417 and comparison with seed HMW-GS using SDS-PAGEM: Protein ladder marker II. In panel A, lanes 1, 3, 5, and 7 show proteins of recombinant plasmid before induction and lanes 2, 4, 6, and 8 show proteins of recombinant plasmid after adding IPTG; the arrows show expression products. In panel B, lane 1 shows the protein of recombinant plasmid before induction; lanes 2, 3, 4, and 5 show proteins of recombinant plasmid KF887414, KF887415, KF887416, and KF887417 after adding IPTG, respectively; lane 6 shows HMW-GS extracted from Dasypyrum villosum seed; the arrows show target proteins.

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图5 KF887414、KF887415、KF887416和KF887417基因的诱导表达产物Western blot检测M: 蛋白质分子质量标准Marker II。1、2、3和4泳道分别为KF887414、KF887415、KF887416和KF887417基因经IPTG诱导后的表达产物; 箭头示基因的表达产物。Fig. 5 Western blotting assay of expressed proteins of genes from KF887414 to KF887417M: Protein ladder marker II; lanes 1, 2, 3, and 4 show proteins of recombinant plasmid KF887414, KF887415, KF887416, and KF887417 after adding IPTG, respectively; the arrows show target proteins.

2.6 目的蛋白的纯化分析

诱导后的蛋白样品经蛋白提取和处理, 用His-Trap HP柱亲和纯化目的蛋白, 回收蛋白溶液, 用12% SDS-PAGE检测, 得到单一条带, 大小与融合表达产物的相似(图6), 表明蛋白纯化成功。

2.7 目的蛋白质的功能鉴定

在4 g基础面粉中添加还原剂DTT和氧化剂KIO₃后, 得到粉质曲线及相应参数(图7-A和表3); 利用氧化还原反应原理, 将纯化后的目的蛋白10 mg加入对照面粉, 得到粉质曲线图(图7-B~E)和相关参数(表3)。与对照相比, 簇毛麦HMW-GS的加入引

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图6 KF887414、KF887415、KF887416和KF887417基因的诱导表达产物柱纯化M: 蛋白质分子质量标准Marker II。1、2、3和4泳道分别为KF887414、KF887415、KF887416和KF887417基因经IPTG诱导后的表达产物; 箭头示基因的表达产物。Fig. 6 Analyses of purification of expressed proteins of genes from KF887414 to KF887417 using SDS-PAGEM: Protein ladder marker II; lanes 1, 2, 3, and 4 show proteins of recombinant plasmid KF887414, KF887415, KF887416, and KF887417 after adding IPTG, respectively; the arrows show target proteins.

起了粉质曲线和粉质参数变化, 其中, DvT、ST、DT和FQN都有不同程度的增加, 并且增幅均存在显著或极显著差异; MTI都有不同程度的下降, 降幅差异显著或极显著; 与对照相比, 加入KF887414和KF887415基因表达产物的DS减小且差异显著, 而加入KF887416和KF887417基因表达产物的DS没有变化; KF887414和KF887415样品的AT时间推后并与对照差异显著, KF887416的AT无变化, 而KF887417的AT提前并且变化极显著(图7、图8和表3)。

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图7 簇毛麦来源的HMW-GS对粉质曲线的影响A: 中国春添加DTT、KIO₃; B: 中国春添加DTT、KIO₃和KF887414; C: 中国春添加DTT、KIO₃和KF887415; D: 中国春添加DTT、KIO₃和KF887416; E: 中国春添加DTT、KIO₃和KF887417。Fig. 7 Effects of HMW-GS from Dasypyrum villosumon farinograph parametersA: Chinese Spring with DTT and KIO₃; B: Chinese Spring with DTT, KIO₃,and KF887414; C: Chinese Spring with DTT, KIO₃,and KF887415; D: Chinese Spring with DTT, KIO₃,and KF887416; E: Chinese Spring with DTT, KIO₃,and KF887417.

表3 纯化蛋白亚基的粉质参数 Table 3 Farinograph parameters of purified HMW-GS from Dasypyrum villosum

表中数据为3次试验的平均值, 均值后大写字母表示0.01差异显著水平。

Data are the average of triplicated micro-mixing experiments. Means marked with different letters are significantly different at P < 0.01. DvT: development times; ST: stability time; DS: degree of time; MTI: mixing tolerance index; AT: arrival time; DT: departure time; FQN: farinograph quality number.

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图8 添加KF887414、KF887415、KF887416和KF887417基因表达蛋白的中国春与中国春(对照)粉质参数的比较DvT: 形成时间; ST: 稳定时间; DS: 弱化度; MTI: 机械耐力系数; AT: 及线时间; DT: 离线时间; FQN: 粉质质量参数。Fig. 8 Comparison of farinogragh parameters between Chinese Spring (control) and Chinese Spring with expressed proteins of KF887414, KF887415, KF887416, and KF887417DvT: development time; ST: stability time; DS: degree of time; MTI: mixing tolerance index; AT: arrival time; DT: departure time; FQN: farinograph quality number.

3 讨论

利用设计的HMW-GS特异引物, 从簇毛麦TA10220基因组中获得43个阳性克隆, 鉴定出6种特异的编码序列。根据编码序列推断, KF887418和KF887419分别在氨基酸第323和第269位处出现终止密码(TGA), 推测可能是由于碱基的转换突变造成的。关于贮藏蛋白编码序列的内部出现终止密码的现象已有报道, 由密码子CAG (Gln)突变成TAG(终止密码)或由CAA (Gln)突变成TAA (终止密码)造成了1Ay、1Ax、1Dx不能正常表达, 成为了假基因, 由于碱基的转换突变引起的基因沉默同样也存在于醇溶蛋白基因中^{[ 30, 31, 32, 33]}。Zhong和Qualset^{[ 34]}通过提取种子全蛋白对122份来源不同的二倍体簇毛麦HMW-GS分析表明, 这些簇毛麦所编码的HMW-GS均在中国春1Dx2亚基和1Dy12亚基之间。理论上这122份簇毛麦的HMW-GS亚基分子量也在70~140 kD之间, 但是该结论未经基因水平的验证, 同时通过提取种子的全蛋白来分析HMW-GS无法排除种子醇溶蛋白和LMW-GS对结论的影响。本研究利用专门分离HMW-GS的方法提取种子HMW-GS, 通过对提取条件的优化尽可能地排除醇溶蛋白和LMW-GS的影响。虽然其余4个真基因的表达蛋白分子量均小于70 kD, 但是体外表达蛋白质和种子来源的HMW-GS具有基本相同的迁移率(图4-B), 表明本研究所得到的4条蛋白质来自簇毛麦TA10220, 并将这6个基因序列根据类型划分为1个y型亚基和5个混合型亚基。显然, 来源于二倍体簇毛麦TA10220的HMW-GS无论在亚基数目及亚基类型上都与普通小麦存在显著差异, 该物种本身的异花授粉特性所导致的高度异质可能对此产生一定的影响^{[ 13, 14]}, 而其分子机制尚需进一步研究。

HMW-GS是影响加工品质重要的遗传因素, 簇毛麦的HMW-GS多为普通小麦不具有的新亚基类型, 并已证实对面筋质量有显著的正向效应。了解簇毛麦HMW-GS基因的分子特征, 对于进一步利用新型HMW-GS基因改良普通小麦面筋质量具有一定的意义。目前已从普通小麦及其亲缘属种中克隆了众多HMW-GS基因, 这些基因的分子量在1.8~2.6 kb之间, 编码的蛋白质分子量在60~140 kD之间(表2)。随着研究的深入以及对结构功能的认识, 发现一些小于1.8 kb的麦谷蛋白基因同样具有HMW-GS的特点^{[ 21, 22, 23, 24, 35, 36, 37, 38, 39, 40, 41, 42]}。本文利用同源克隆技术克隆到的簇毛麦HMW-GS基因小于1.8 kb, 但其推导氨基酸序列, 具有HMW-GS结构特点(图2), 编码区没有内含子, 具有21个氨基酸残基的信号肽, 高度保守的N-和C-端及中间重复区^{[ 11, 12]}, 尽管分子量较小, 也应该是HMW-GS基因。这与前人所克隆的簇毛麦HMW-GS基因结果^{[ 20, 21, 22, 23, 24]}较为一致。由于簇毛麦与小麦的亲缘关系较远, 传统的以大小区分HMW-GS的方法, 在簇毛麦研究分析上也许存在一定的局限性。普通小麦HMW-GS大小的分类方法不适合对远缘材料的分类(表2), 即不能仅仅依据SDS- PAGE上的迁移率区分。尤其在对利用簇毛麦等远缘材料所创制的材料, 包括易位系、附加系鉴定的时候, 不能仅仅靠蛋白质分子量大小进行染色体易位片段的判断。

本研究得到的所有簇毛麦HMW-GS基因编码的氨基酸序列, N-端保守区有5个半胱氨酸残基, 并且第3和第4位半胱氨酸残基相邻, 与报道的y型HMW-GS的N-端保守区的特征一致(图2和表2); N-进化树分析也表明该6条推导的氨基酸序列的N-端保守区与y型关系较近与x型关系较远(图3-B)。KF887414的中间重复区六肽(PGQGQQ)和九肽(GYYPTSP/LQQ)高度重复, 不存在三肽(GQQ), 表明KF887414基因编码的氨基酸序列具有y型HMW-GS的中间重复区的特征^{[ 2, 3]}, 而KF887415~ KF887419除重复出现六肽(PGQGQQ)和九肽(GYYPTSP/LQQ)外, 还具有类似三肽(GQQ)PGQ, 表明这些序列的中间重复区具有典型的x型HMW-GS特征^{[ 2, 3]}, 庞玉辉等^{[ 21]}报道的N-端保守区具有y型HMW-GS特征的簇毛麦高分子谷蛋白的中间重复区也存在PGQ、GQQ和RGQ等三肽。本研究中, 氨基酸序列的C-端保守区具有一个半胱氨酸残基。C-端进化树分析表明KF887414与y型亲缘关系较近, 而KF887415~KF887419与x型关系较近(图3-C)。KF887414的近C-端重复区多1个半胱氨酸残基, KF887419的近N-端重复区多出1个(图2)。以上分析表明, 某些簇毛麦HMW-GS兼有普通小麦来源的x和y型的一些结构特征, 此种现象可能是簇毛麦物种本身的特异性所致。因此, 用以区分栽培小麦x和y型HMW-GS的分类方法需要进行适当完善方能反映其亲缘关系较远的野生种属所具有的同源亚基的属性。

利用微量掺粉试验可以直观地检测亚基的品质效应, 簇毛麦TA10220来源的4个HMW-GS的掺入均使整个面团的空间结构经历了某些剧烈改变, 并最终表现为粉质曲线图的明显变化(图7): 面团的形成时间延长, 表明上述亚基具有提高基础面粉面筋质量的潜力; 稳定时间延长表明提高了面团的耐揉性、韧性和面筋的强度; 离线时间延长表明面筋强度或耐受过度搅拌的能力增强; 粉质质量参数显著提高表明面团的综合质量具有显著的正向效应; 耐搅拌指数降低说明面团的耐揉性提高(图8和表3)。因此, 簇毛麦中的HMW-GS对面团的品质有正向效应。通过杂交育种的方式将簇毛麦的HMW-GS基因转入小麦, 对改良普通小麦的加工品质, 增加其遗传多样性具有重要意义。

4 结论

克隆到6个HMW-GS基因序列, 均小于1.8 kb, 其中KF887418和KF887419是编码区具有终止信号的假基因, 这6个序列均具有HMW-GS显著的结构特征, 并且与已知的HMW-GS的亲缘关系较近; 其中KF887414为y型HMW-GS, 而其他序列均同时具有x和y型HMW-GS结构特征。簇毛麦来源的HMW-GS对面粉的加工品质有显著的正向效应。

The authors have declared that no competing interests exist.

作者已声明无竞争性利益关系。The authors have declared that no competing interests exist.

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[15]	De Pace C, Snidaro D, Ciaffi M, Vittori D, Ciofo A, Cenci A, Tanzarella O A, Qualset C O, Scarascia Mugnozza G T. Introgression of Dasypyrum villosum chromatin into common wheat improves grain protein quality. Euphytica, 2001, 117: 67-75 [本文引用:2] [JCR: 1.643]
[16]	Zhao W C, Qi L L, Gao X, Zhang G S, Dong J, Chen Q J, Bernd F, Bikram S G. Development and characterization of two new Triticum aestivum-Dasypyrum villosum Robertsonian translocation lines T1DS·1V#3L and T1DL·1V#3S and their effect on grain quality. Euphytica, 2010, 175: 343-350 [本文引用:2] [JCR: 1.643]
[17]	董剑, 杨华, 赵万春, 李晓燕, 陈其皎, 高翔. 普通小麦中国春-簇毛麦异位系T1DL·1VS和T1DS·1VL的农艺和品质特性. 作物学报, 2013, 39: 1386-1390 Dong J, Yang H, Zhao W C, Li X Y, Chen Q J, Gao X. Agronomic traits and grain quality of Chinese Spring-Dasypyrum villosum translocation lines T1DL·1VS and T1DS·1VL. Acta Agron Sin, 2013, 39: 1386-1390 (in Chinese with English abstract) [本文引用:2] [CJCR: 1.667]
[18]	Montebove L, De Pace C, Jan C C, Scarascia Mugnozza G T, Qualse C O. Chromosomal location of isozyme and seed storage protein genes in Dasypyrum villosum (L. ) Cand argy. Theor Appl Genet, 1987, 73: 836-845 [本文引用:2] [JCR: 3.658]
[19]	Shewry P R, Parmar C, Pappin D C. Characterization and genetic control of the prolamins of Haynaldia villosa: relationship to cultivated species of the Triticeae (rye, wheat, and barley). Biochem Genet, 1987, 25: 309-325 [本文引用:1] [JCR: 0.938]
[20]	张瑞奇. 簇毛麦籽粒硬度基因和贮藏蛋白基因的染色体定位及易位系选育. 南京农业大学博士学位论文, 2010 Zhang R Q. Chromosome Location of Genes for Hardness Locus and Storage Proteins in H. villosa and Creation the Translocation Lines. PhD Dissertation of Nanjing Agricultural University, Nanjing, China, 2010 (in Chinese with English abstract) [本文引用:3]
[21]	庞玉辉, 陈新宏, 赵继新, 武军, 程雪妮, 刘淑会, 杨群慧, 杜万里, 陈林刚. 簇毛麦HMW-GS及其启动子基因的克隆与序列分析. 西北植物学报, 2009, 29: 859-866 Pang Y H, Chen X H, Zhao J X, Wu J, Cheng X N, Liu S H, Yang Q H, Du W L, Chen L G. Cloning and sequence analysis of the HMW-GS gene and its promoter from Dasypyrum villosum. Acta Bot Boreali-Occident Sin, 2009, 29: 859-866 (in Chinese with English abstract) [本文引用:4] [CJCR: 1.321]
[22]	陈凡国, 朱翔宇, 夏光敏. 簇毛麦中一种新型高分子量麦谷蛋白亚基基因序列的研究. 西北植物学报, 2005, 25: 1410-1414 Chen F G, Zhu X Y, Xia G M. Sequence of a new HMW glutenin subunit gene in Haynaldia villosa. Acta Bot Boreali-Occident Sin, 2005, 25: 1410-1414 (in Chinese with English abstract) [本文引用:3] [CJCR: 1.321]
[23]	刘守斌. 簇毛麦染色体分子标记的筛选及其高分子量谷蛋白亚基基因的克隆. 中国农业大学博士学位论文, 2002 Liu S B. The Characterization of V-Genome Specific Molecular Marker and Cloning of High-Molecular-Weight Glutenin Subunit Genes in Haynaldia villosa. PhD Dissertation of China Agricultural University, Beijing, China, 2002 (in Chinese with English abstract) [本文引用:3]
[24]	陈晓燕. 簇毛麦储藏蛋白基因的克隆及小麦的抗白粉病育种. 山东大学硕士学位论文, 2009 Chen X Y. Glutenin and Gliadin Genes Cloning from Dasypyrum villosum and Powdery Mildew Resistance Breeding in Wheat. MS Thesis of Shand ong University, Tai’an, Shand ong, China, 2009 (in Chinese with English abstract) [本文引用:3]
[25]	段淑娥, 赵文明. 小麦谷蛋白亚基的快速提取分离及SDS-PAGE分析. 陕西师范大学学报(自然科学版), 2004, 32(1): 77-79 Duan S E, Zhao W M. Rapid separation and SDS-PAGE analysis of wheat glutenin subunits. J Shaanxi Normal Univ (Nat Sci Edn), 2004, 32(1): 77-79 (in Chinese with English abstract) [本文引用:2]
[26]	Verbruggen I M, Veraverbeke W S, Vand amme A, Delcour A. Simultaneous isolation of wheat high molecular weight and low molecular weight glutenin subunits. J Cereal Sci, 1998, 28: 25-32 [本文引用:2] [JCR: 2.088]
[27]	Murray M G, Thompson W F. Rapid isolation of high molecular weight plant DNA. Nucl Acids Res, 1980, 8: 4321-4325 [本文引用:1]
[28]	Xu H, Wang R J, Shen X, Zhao Y L, Sun G L, Zhao H X, Guo A G. Functional properties of a new low-molecular-weight glutenin subunit gene from a bread wheat cultivar. Theor Appl Genet, 2006, 113: 1295-1303 [本文引用:1] [JCR: 3.658]
[29]	Chen F G, Zhao F, Liu R K, Xia G M. Functional properties of two low-molecular-weight glutenin subunits carrying additional cysteine residues from hybrid introgression line II-12 derived from Triticum aestivum and Agropyron elongatum. Food Chem, 2011, 127: 1773-1776 [本文引用:1] [JCR: 3.334]
[30]	Forde J, Malpica J M, Halford N G, Shewry P R, Anderson O D, Greene F C, Miflin B J. The nucleotide sequence of a HMW glutenin subunit gene located on chromosome 1A of wheat (Triticum aestivum L. ). Nucl Acids Res, 1985, 13: 6817-6832 [本文引用:1]
[31]	De Bustos A, Rubio P, Jouve N. Molecular characterisation of the inactive allele of the gene Glu-A1 and the development of a set of AS-PCR markers for HMW glutenins of wheat. Theor Appl Genet, 2000, 100: 1085-1094 [本文引用:1] [JCR: 3.658]
[32]	Wan Y, Wang D, Shewry P, Halford N. Isolation and characterization of five novel high molecular weight subunit of glutenin genes from Triticum timopheevi and Aegilops cylindrica. Theor Appl Genet, 2002, 104: 828-839 [本文引用:1] [JCR: 3.658]
[33]	Anderson O D, Greene F C. The characterization and comparative analysis of high-molecular-weight glutenin genes from genomes A and B of a hexaploid bread wheat. Theor Appl Genet, 1989, 77: 689-700 [本文引用:1] [JCR: 3.658]
[34]	Zhong G Y, Qualset C O. Allelic diversity of high-molecular- weight glutenin protein subunits in natural populations of Dasypyrum villosum (L. ) Cand argy. Theor Appl Genet, 1993, 86: 851-858 [本文引用:1] [JCR: 3.658]
[35]	Feng D S, Chen F G, Zhao S Y, Xia G M. High-molecular-weight glutenin subunit genes in decaploid Agropyron elongatum. Acta Bot Sin, 2004, 46: 489-496 [本文引用:1] [JCR: 0.599]
[36]	封德顺, 陈凡国, 赵双宜, 夏光敏. 高冰草中一种新型高分子量谷蛋白亚基编码序列的研究. 西北植物学报, 2004, 24: 237-242 Feng D S, Chen F G, Zhao S Y, Xia G M. Study on a novel HMW glutenin subunit coding region from Agropyron elongatum. Acta Bot Boreali-Occident Sin, 2004, 24: 237-242 (in Chinese with English abstract) [本文引用:1] [CJCR: 1.321]
[37]	Wang J R, Yan Z H, Wei Y M, Zheng Y L. A novel high- molecular-weight glutenin subunit gene Ee1. 5 from Elytrigia elongate (Host) Nevski. J Cereal Sci, 2004, 40: 289-294 [本文引用:1] [JCR: 2.088]
[38]	Wang J R, Yan Z H, Wei Y M, Zheng Y L. Characterization of high-molecular-weight glutenin subunit genes from Elytrigia elongata. Plant Breed, 2006, 125: 89-95 [本文引用:1] [JCR: 1.175]
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[40]	Liu S W, Zhao S Y, Chen F G, Xia G M. Generation of novel high quality HMW-GS genes in two introgression lines of Triticum aestivum/Agropyron elongatum. BMC Evol Biol, 2007, 7: 76 [本文引用:1] [JCR: 3.285]
[41]	Liu S W, Gao X, Xia G M. Characterizing HMW-GS alleles of decaploid Agropyron elongatum in relation to evolution and wheat breeding. Theor Appl Genet, 2008, 116: 325-334 [本文引用:1] [JCR: 3.658]
[42]	Jiang Q T, Wei Y M, Lu Z X, Pu Z E, Lan X J, Zheng Y L. Structural variation and evolutionary relationship of novel HMW glutenin subunits from Elymus glaucus. Hereditas, 2010, 147: 136-141 [本文引用:1] [CJCR: 0.928]

1996

38.597

0.0

... 小麦高分子量谷蛋白亚基(HMW-GS)和低分子量谷蛋白亚基(LMW-GS)通过分子内和分子间二硫键形成聚合蛋白, 从而影响面粉的加工品质^[1] ...

1987

0.0

... HMW-GS影响面团的弹性和面包的加工品质^[2,3] ...

... HMW-GS典型的结构由4部分组成, 分别是信号肽(蛋白质加工过程被删除)、高度保守的N-端和C-端以及中间重复区^[2,3] ...

... x和y型HMW-GS主要区别在于半胱氨酸残基的个数和位置, 中间重复区重复单元的类型, 其中六肽以PGQGQQ、九肽以GYYPTS/PLQQ的保守形式出现在所有x、y型亚基中, 而三肽以GQQ的保守形式只存在在x型亚基中^[2,3] ...

... KF887414的中间重复区六肽(PGQGQQ)和九肽(GYYPTSP/LQQ)高度重复, 不存在三肽(GQQ), 表明KF887414基因编码的氨基酸序列具有y型HMW-GS的中间重复区的特征^[2,3], 而KF887415~ KF887419除重复出现六肽(PGQGQQ)和九肽(GYYPTSP/LQQ)外, 还具有类似三肽(GQQ)PGQ, 表明这些序列的中间重复区具有典型的x型HMW-GS特征^[2,3], 庞玉辉等^[21]报道的N-端保守区具有y型HMW-GS特征的簇毛麦高分子谷蛋白的中间重复区也存在PGQ、GQQ和RGQ等三肽 ...

1992

2.088

0.0

... HMW-GS影响面团的弹性和面包的加工品质^[2,3] ...

... HMW-GS典型的结构由4部分组成, 分别是信号肽(蛋白质加工过程被删除)、高度保守的N-端和C-端以及中间重复区^[2,3] ...

... 但是1By9、1Dy10和1Dy12近C-端的中间重复区多了1个半胱氨酸残基, 而1Dx5近N-端的中间重复区多了1个半胱氨酸残基^[3] ...

1980

0.0

... 编码HMW-GS (60~120 kD)的基因位于第1部分同源群染色体1A、1B和1D长臂上^{[4,5,6,7,8,9]}, 每个位点由2个紧密连锁的基因组成, 其中较大的基因编码x型HMW-GS的合成, 较小的基因编码y型HMW-GS的合成^[9,10] ...

1981

3.658

0.0

1980

3.658

0.0

1982

3.658

0.0

1987

1.759

0.0

1996

2.088

0.0

1986

0.938

0.0

1989

0.0

... 端序列, 编码区内部不存在内含子^[11,12] ...

... 8 kb, 但其推导氨基酸序列, 具有HMW-GS结构特点(图2), 编码区没有内含子, 具有21个氨基酸残基的信号肽, 高度保守的N-和C-端及中间重复区^[11,12], 尽管分子量较小, 也应该是HMW-GS基因 ...

1995

3.658

0.0

... 端序列, 编码区内部不存在内含子^[11,12] ...

2006

1.643

0.0

. 2006, 152:429-440 DOI:10.1007/s10681-006-9232-2

The genus Dasypyrum: 1. The taxonomy and relationships within Dasypyrum and with Triticeae species

1.Agricultural University of Lublin Insitute of Genetics and Plant Breeding 20-950 Lublin ul Akademicka 13 Poland

The article is an attempt to summarize available information about the relationships within Dasypyrum (Triticeae, Poaceae) and between Dasypyrum and other genera in the Triticeae. A relationship between the annual diploid D. villosum (L.) P. Candargy and the perennial tetraploid D. breviaristatum (L.) Frederiksen is discussed on the basis of cytological and molecular investigations. Although the relationship between the two Dasypyrum species seems to be quite close, D. breviaristatum is an autotetraploid with a basic genome different from that of D. villosum. The position of Dasypyrum in relation to other Triticeae is summarized, with special attention being paid to the Triticum and Secale. The closer relationship of Dasypyrum with Secale in comparison to Triticum seems to be reliable, even if only few intergeneric hybrids have been produced. It may be related to the fact that only a few experiments on production of those hybrids have been conducted till the present day, in contrast to experiments concerning hybrids with Triticum.

... 簇毛麦(Dasypyrum villosum, 2n = 14, VV)属于禾本科小麦亚族簇毛麦属, 为一年生二倍体异花授粉草本植物, 具有抗多种病害、品质优良、抗旱耐寒、密穗、多花及分蘖能力强等特性, 还带有提高籽粒蛋白质含量、赖氨酸含量和面筋强度的基因, 是普通小麦遗传改良优异的三级基因源^[13,14] ...

... 显然, 来源于二倍体簇毛麦TA10220的HMW-GS无论在亚基数目及亚基类型上都与普通小麦存在显著差异, 该物种本身的异花授粉特性所导致的高度异质可能对此产生一定的影响^[13,14], 而其分子机制尚需进一步研究 ...

2006

1.643

0.0

. 2006, 152:441-454 DOI:10.1007/s10681-006-9245-x

The genus Dasypyprum: 2. Dasypyrum villosum: a wild species used in wheat improvement

1.Insitute of Genetics and Plant Breeding, Agricultural University of Lublin, 20-950 Lublin, Ul Akademicka 13, Poland

Dasypyrum villosum (L.) P. Candargy is discussed as a species commonly used in wheat improvement. Chromosomal localization of the potentially useful traits and chromosomal position of some morphological and isozyme markes are shown. The investigations using molecular RAPD, AFLP, SSR, RFLP markers and in situ (GISH, FISH) hybridization experiments on D. villosum itself and in wide hybrids with Triticum are summarized. The article also presents the information about designation of D. villosum chromosomes and the current knowledge on the phenomenon of two-coloured D. villosum caryopses.

2001

1.643

0.0

. 2001, 117:67-75 DOI:10.1023/A:1004095705460

Introgression of Dasypyrum villosum chromatin into common wheat improves grain protein quality

1.University of Tuscia 2.University of California

Dasypyrum villosum (L.) Candargy (DV) is adiploid (2n = 14, VV genomes), allogamous grass of theMediterranean region. It may be hybridized with wheatand is thus a gene resource for wheat improvement. Westudied grain protein concentration andSDS-sedimentation (SED) as indicators of end-usequality. The latter is a good predictor of glutenstrength. A-PAGE and SDS-PAGE were used to identifymonomeric and polymeric seed storage proteins,respectively, to relate proteins of DV to those foundin Chinese Spring (CS), Triticum aestivum L.,wheat. Two full-sib lines of DV had high grain protein(19.3 and 20.3%), but one had very low mean SED (69mm) and one had very high (118 mm) based on onegreenhouse and one field test. CS had very low grainprotein (12.0%) and weak gluten (33 mm). Single-DVchromosome addition and substitution lines and twoDV-wheat recombinant lines all had higher grainprotein than CS (range 13.9 to 16.7%). SED valuesshowed a different pattern. CS=4V and CS=6V hadlow SED, 63 and 44 mm, similar to CS, whereas CS=1Vand full sib DV 200 had very strong gluten, 118 mm, asdid substitution lines CS cdot

1V (1A) and CS cdot

1V (1B), 125and 131 mm, respectively. One hybrid-derived line withDV-wheat 1V recombinant chromosome had SED of 99 mmand one line with a 6V added chromosome had SED of 64mm. The large positive effects of quality in the wheathaving DV chromosome 1V are believed to be due to DValleles at the Glu-V1 and Gli-V1/Glu-V3loci. DV chromosomes 4V and 6V did not contribute toimproved quality probably due to Gli-V2 and Gli-V3 which, as the orthologous loci in wheat, donot enhance wheat quality. Based on the positiveeffects of alleles on DV chromosome 1V in a breadwheat background, we conclude that D. villosumis a source of allelic diversity that can beconsidered for improving end-use quality in breadwheat.

... 簇毛麦1V染色体对小麦加工品质具有正效应, 可以显著提高面筋的强度, 是位于1V染色体上Glu-V1、Gli-V1和Glu-V13位点的作用^{[15,16,17,18]} ...

... 型)醇溶蛋白基因Gli-V1位点和编码低分子谷蛋白Glu-V3位点^{[15,16,17,18,19,20]} ...

2010

1.643

0.0

. 2010, 175:343-350 DOI:10.1007/s10681-010-0177-0

Development and characterization of two new Triticum aestivum-Dasypyrum villosum Robertsonian translocation lines T1DS#cod#x000b7;1V#3L and T1DL#cod#x000b7;1V#3S and their effect on grain quality

1.College of Agronomy, Northwest Agriculture & Forestry University, Yangling, Shaanxi 712100, China 2.USDA-ARS, Northern Crop Science Laboratory, Fargo, ND 58105-5677, USA 3.Department of Plant Pathology, Wheat Genetic and Genomic Resources Center, Throckmorton Plant Sciences Center, Kansas State University, Manhattan, KS 66506, USA

Dasypyrum villosum (L.) Candargy is a diploid, wild relative of bread wheat (Triticum aestivum L.). Previous studies showed that D. villosum chromosome 1V has genes that encode seed storage proteins that may be used to enhance the grain quality of bread wheat. As a first step in genetic transfer, the present study was initiated to develop compensating Robertsonian translocations involving wheat chromosome 1D and D. villosum chromosome 1V and to analyze their effects on grain quality. A monosomic 1D stock was crossed with the disomic addition stock DA1V#3 and the double monosomic plants (20″ + 1D′ + 1V#3′) were self pollinated. Two co-dominant STS markers (BE499250 and BE591682) polymorphic for the short arm of 1V#3S and two dominant STS markers (BE518358 and BE585781) polymorphic for the long arm of 1V#3L were developed to screen a large number of progeny to identify plants that had only the 1V#3S or 1V#3L arms. Five compensating Robertsonian heterozygous translocations, two (plants #56 and #83) for the short arm (T1DL·1V#3S) and three (plants #7, #123, and #208) for the long arm (T1DS·1V#3L) were identified from 282 F₂ plants and confirmed by genomic in situ hybridization and C-banding analyses. Two homozygous translocations T1DL·1V#3S (plants #14 and #39) were identified from 52 F₃ plants derived from F₂ plant #83. Four homozygous translocations T1DS·1V#3L (plants #3, #22, #29, and #30) were identified from 68 F₃ plants derived from F₂ plant #208. The homozygous translocation T1DL·1V#3S had a significantly higher (37.4 ml) and T1DS·1V#3L had significantly lower (10 ml) Zeleny sedimentation values compared to Chinese Spring wheat (30.7 ml). Our results showed that 1V#3S increased gluten strength and enhanced wheat quality, but 1V#3L decreased gluten strength and did not enhance wheat quality.

... 簇毛麦1V染色体对小麦加工品质具有正效应, 可以显著提高面筋的强度, 是位于1V染色体上Glu-V1、Gli-V1和Glu-V13位点的作用^{[15,16,17,18]} ...

... 型)醇溶蛋白基因Gli-V1位点和编码低分子谷蛋白Glu-V3位点^{[15,16,17,18,19,20]} ...

2013

0.0

1.667

. 2013, 39:1386-1390 DOI:10.3724/SP.J.1006.2013.01386

Agronomic traits and grain quality of Chinese Spring-Dasypyrum villosum translocation lines T1DL#cod#x000b7;1VS and T1DS#cod#x000b7;1VL

(普通小麦中国春-簇毛麦异位系T1DL#cod#x000b7;1VS和T1DS#cod#x000b7;1VL的农艺和品质特性) . (作物学报) , 2013 , 39 : 1386 - 1390 Dong J , Yang H , Zhao W C , Li X Y , Chen Q J , Gao X . Agronomic traits and grain quality of Chinese Spring-Dasypyrum villosum translocation lines T1DL#cod#x000b7;1VS and T1DS#cod#x000b7;1VL

Dong J

(董剑) , (杨华) , (赵万春) , (李晓燕) , (陈其皎) , (高翔) . (普通小麦中国春-簇毛麦异位系T1DL#cod#x000b7;1VS和T1DS#cod#x000b7;1VL的农艺和品质特性) . (作物学报) , 2013 , 39 : 1386 - 1390 Dong J

赵万春, E-mail:zhaowc2009@hotmail.com

To understand the effect on agronomic traits and grain processing quality of Chinese Spring (CS)–Dasypyrum villosa compensating Robertsonian translocation lines T1DL?1VS and T1DS?1VL, we conducted a 2-year field trial at Yangling, Shaanxi Province from 2008 to 2011. Some agronomic traits and processing quality characteristics of T1DL?1VS and T1DS?1VL were compared with those of CS, and the high-molecular-weight glutein subunit (HMW-GS) genes of D. villosum were located on chromosome arms using SDS-PAGE method. The heading and maturity dates of both translocation lines were the same, but 1–2 d later than those of CS. Compared to CS, T1DS?1VL had similar other agronomic traits; however, T1DL?1VS showed higher values of spring tillers per plant, 1000-grain weight, and grain weight per plant. Although the seed protein contents of both translocation lines were not significantly different from those of CS, T1DS?1VL exhibited significantly lower Zeleny sedimentation value, developing time, stability, and quality index of farinograph than CS; the values of these quality parameters were significantly higher in T1DL?1VS than in CS. Therefore, T1DS?1VL has a significant negative effect on wheat dough strength, and T1DL?1VS significantly increase gluten strength. HMW-GS gene Glu-V1 of D. villosum was located on both of 1VS and 1VL, but the gene expression level might be higher in T1DL?1VS than in T1DS?1VL.

为明确普通小麦中国春–簇毛麦整臂互补易位系T1DS?1VL和T1DL?1VS对农艺特性和加工品质的效应, 于2008—2010年在陕西杨凌连续2个生长季对这2个易位系和CS的主要农艺性状和加工品质性状进行了研究, 并采用SDS-PAGE法对簇毛麦高分子量麦谷蛋白亚基(HMW-GS)基因进行了进一步的染色体臂定位。结果表明, 这2个易位系的抽穗期和成熟期相同, 但比CS晚1~2 d, T1DS?1VL其他农艺性状与中国春相似, T1DL?1VS的春季单株分蘖、千粒重和单株粒重显著高于中国春；2个易位系的籽粒蛋白质含量与中国春无显著差异, T1DS?1VL的Zeleny沉淀值、面团形成时间、稳定时间和粉质仪质量指数显著降低；相反, T1DL?1VS的这些性状值显著提高。说明T1DS?1VL易位系对小麦的面团强度有显著的负向效应, 而T1DL?1VS易位系显著增强面筋强度。SDS-PAGE分析结果表明, 簇毛麦的1VS和1VL上均含有簇毛麦的HMW-GS基因Glu-V1, 但该基因在T1DL?1VS中的表达可能强于在T1DS?1VL中。

... 簇毛麦1V染色体对小麦加工品质具有正效应, 可以显著提高面筋的强度, 是位于1V染色体上Glu-V1、Gli-V1和Glu-V13位点的作用^{[15,16,17,18]} ...

... 型)醇溶蛋白基因Gli-V1位点和编码低分子谷蛋白Glu-V3位点^{[15,16,17,18,19,20]} ...

1987

3.658

0.0

... 簇毛麦1V染色体对小麦加工品质具有正效应, 可以显著提高面筋的强度, 是位于1V染色体上Glu-V1、Gli-V1和Glu-V13位点的作用^{[15,16,17,18]} ...

... 型)醇溶蛋白基因Gli-V1位点和编码低分子谷蛋白Glu-V3位点^{[15,16,17,18,19,20]} ...

1987

0.938

0.0

... 型)醇溶蛋白基因Gli-V1位点和编码低分子谷蛋白Glu-V3位点^{[15,16,17,18,19,20]} ...

2010

0.0

... 型)醇溶蛋白基因Gli-V1位点和编码低分子谷蛋白Glu-V3位点^{[15,16,17,18,19,20]} ...

... 利用同源克隆技术, 一些研究者从簇毛麦中分离到HMW-GS编码基因及其启动子序列, 并发现了没有功能的假基因^{[20,21,22,23,24]} ...

... 这与前人所克隆的簇毛麦HMW-GS基因结果^{[20,21,22,23,24]}较为一致 ...

2009

0.0

1.321

. 2009, 29:859-866

Cloning and sequence analysis of the HMW-GS gene and its promoter from Dasypyrum villosum

(簇毛麦HMW-GS及其启动子基因的克隆与序列分析) . (西北植物学报) , 2009 , 29 : 859 - 866 Pang Y H , Chen X H , Zhao J X , Wu J , Cheng X N , Liu S H , Yang Q H , Du W L , Chen L G . Cloning and sequence analysis of the HMW-GS gene and its promoter from Dasypyrum villosum

Pang Y H

(庞玉辉) , (陈新宏) , (赵继新) , (武军) , (程雪妮) , (刘淑会) , (杨群慧) , (杜万里) , (陈林刚) . (簇毛麦HMW-GS及其启动子基因的克隆与序列分析) . (西北植物学报) , 2009 , 29 : 859 - 866 Pang Y H

利用2对特异引物,从簇毛麦(Dasypyrum villosum)基因组中分离克隆出一个簇毛麦HMW-GS基因VHG-2(GenBank登录号为FJ600492)及其启动子序列VHGp-1(GenBank登录号为FJ600489).VHGp-1序列长度为1 099 bp,从5′至3′方向依次有E-box、N-box、G-box、HMW谷蛋白特异38 bp增强子和TATA-box等典型的HMW-GS基因启动子作用调控元件,说明VHGp-1为簇毛麦HMW-GS的启动子基因.VHG-2序列长度为1 572 bp,具有单一完整的、可编码498个氨基酸的开放阅读框(ORF),该ORF推导的氨基酸序列结构分析表明,编码区依次包含由21个氨基酸残基组成的信号肽、105个氨基酸残基组成的N-末端区、330个氨基酸残基组成的中部重复区和42个氨基酸残基组成的C-末端区;中部重复区主要重复单元为6肽(PQQGQQ)和9肽(GYYPTSP/LQQ);有6个半胱氨酸残基(Cys),其中5个分布在N-末端区,1个分布在C-末端区,第3、4个相邻.这些特征与报道的y-型HMW-GS多肽结构基本一致,说明VHG-2是簇毛麦的y-型HMW-GS基因.系统进化分析表明,簇毛麦HMW-GS启动子序列(VHGP-1)与智利大麦(H.chilense)H基因组的D-hordein基因、拟鹅观草和阿拉善鹅观草St基因组的HMW-GS基因的启动子具有比较近的同源关系,簇毛麦HMW-GS基因(VHG-2)与冰草、拟鹅观草和中间偃麦草的y-型HMW-GS基因具有较近的同源关系.

... 利用同源克隆技术, 一些研究者从簇毛麦中分离到HMW-GS编码基因及其启动子序列, 并发现了没有功能的假基因^{[20,21,22,23,24]} ...

... 8 kb的麦谷蛋白基因同样具有HMW-GS的特点^{[21,22,23,24,35,36,37,38,39,40,41,42]} ...

... 这与前人所克隆的簇毛麦HMW-GS基因结果^{[20,21,22,23,24]}较为一致 ...

2005

0.0

1.321

. 2005, 25:1410-1414

Sequence of a new HMW glutenin subunit gene in Haynaldia villosa

(簇毛麦中一种新型高分子量麦谷蛋白亚基基因序列的研究) . (西北植物学报) , 2005 , 25 : 1410 - 1414 Chen F G , Zhu X Y , Xia G M . Sequence of a new HMW glutenin subunit gene in Haynaldia villosa

Chen F G

(陈凡国) , (朱翔宇) , (夏光敏) . (簇毛麦中一种新型高分子量麦谷蛋白亚基基因序列的研究) . (西北植物学报) , 2005 , 25 : 1410 - 1414 Chen F G

簇毛麦(Haynaldia villosa)是普通小麦品质改良的重要亲本之一.利用基因组PCR的方法从簇毛麦中克隆到一个新的高分子量麦谷蛋白亚基(HMW-GS)基因(HayviG1)全编码序列,内有2个终止密码,可能是1个假基因.从推导的氨基酸序列同源性分析表明:HayviG1编码1个新的HMW-GS亚基,聚类分析表明与长穗偃麦草的Agelog5以及圆柱山羊草的1Cy具有较近的同源性.

... 利用同源克隆技术, 一些研究者从簇毛麦中分离到HMW-GS编码基因及其启动子序列, 并发现了没有功能的假基因^{[20,21,22,23,24]} ...

... 8 kb的麦谷蛋白基因同样具有HMW-GS的特点^{[21,22,23,24,35,36,37,38,39,40,41,42]} ...

... 这与前人所克隆的簇毛麦HMW-GS基因结果^{[20,21,22,23,24]}较为一致 ...

2002

0.0

... 利用同源克隆技术, 一些研究者从簇毛麦中分离到HMW-GS编码基因及其启动子序列, 并发现了没有功能的假基因^{[20,21,22,23,24]} ...

... 8 kb的麦谷蛋白基因同样具有HMW-GS的特点^{[21,22,23,24,35,36,37,38,39,40,41,42]} ...

... 这与前人所克隆的簇毛麦HMW-GS基因结果^{[20,21,22,23,24]}较为一致 ...

2009

0.0

... 利用同源克隆技术, 一些研究者从簇毛麦中分离到HMW-GS编码基因及其启动子序列, 并发现了没有功能的假基因^{[20,21,22,23,24]} ...

... 8 kb的麦谷蛋白基因同样具有HMW-GS的特点^{[21,22,23,24,35,36,37,38,39,40,41,42]} ...

... 这与前人所克隆的簇毛麦HMW-GS基因结果^{[20,21,22,23,24]}较为一致 ...

2004

0.0

. 2004, 32(1):77-79

Rapid separation and SDS-PAGE analysis of wheat glutenin subunits

(小麦谷蛋白亚基的快速提取分离及SDS-PAGE分析) . (陕西师范大学学报(自然科学版) ), 2004 , 32 ( 1 ): 77 - 79 Duan S E , Zhao W M . Rapid separation and SDS-PAGE analysis of wheat glutenin subunits

Duan S E

(段淑娥) , (赵文明) . (小麦谷蛋白亚基的快速提取分离及SDS-PAGE分析) . (陕西师范大学学报(自然科学版) ), 2004 , 32 ( 1 ): 77 - 79 Duan S E

用70%乙醇和50%异丙醇除去醇溶蛋白,以二硫苏糖醇(DTT)为还原剂,在50%异丙醇,0.08 mol/L Tris-HCl(pH为8.0)缓冲液中提取总麦谷蛋白亚基,加入丙酮至浓度为40%时,高分子量麦谷蛋白亚基(HMW-GS)沉淀析出;继续加入丙酮至80%时,析出低分子量麦谷蛋白亚基(LMW-GS).在3.3%浓缩胶和10%分离胶的不连续体系中进行SDS-PAGE电泳,结果表明,该方法可以有效地除去醇溶蛋白对麦谷蛋白亚基电泳分析的背景干扰,HMW-GS和LMW-GS的提取、分离一步完成,操作简便,时间短,对大量小麦样品的HMW-GS和LMW-GS可以快速分离、提纯和鉴定.

... 1 HMW-GS提取及SDS-PAGE分析参考段淑娥等^[25]和Verbruggen等^[26]的方法, 从单粒种子中提取簇毛麦TA10220 (美国堪萨斯州大学小麦遗传资源研究中心提供) HMW-GS, 然后进行SDS-PAGE分析^[25,26] ...

1998

2.088

0.0

1980

0.0

... 2 目的基因的克隆与序列分析以室内培养2周的簇毛麦TA10220幼嫩叶片为材料, 采用微量CTAB法提取基因组DNA^[27] ...

2006

3.658

0.0

. 2006, 113:1295-1303 DOI:10.1007/s00122-006-0383-2

Functional properties of a new low-molecular-weight glutenin subunit gene from a bread wheat cultivar

1.College of Life Sciences, Northwest A & F University, Yangling, Shaanxi 712100, People’s Republic of China 2.Department of Biology, Saint Mary’s University, Halifax, N S Canada B3H 3C3 3.Key Laboratory of Agricultural Molecular Biology of Shannxi Province, Yangling, Shaanxi 712100, People’s Republic of China

Some allelic forms of low-molecular-weight glutenin subunit (LMW-GS) can greatly influence the end-use of wheat flours, understanding the function of each allele of LMW-GS is important to wheat quality breeding. A LMW-GS gene XYGluD3-LMWGS 1(AY263369) has been cloned from bread wheat cultivar Xiaoyan 6. The deduced protein contained nine cystine residues, one more than that in all other LMW-GSs reported previously, indicating that it is either a new gene or a new allele of a known LMW-GS gene. In this study, the gene was expressed in E. coil in large scale for the testing of its functional property. Reactive Red 120-Agarose resin was used efficiently to purify the expressed LMW-GS proteins from bacteria, with the lactic acid–sodium lactate buffer (pH 4.5) which contained low concentration SDS as elution solution. The purified protein (belonging to the LMW-m family, MW about 35 KDa) was supplemented into a base flour, the results of 10 g dough mixing test indicated that incorporation of the LMW-GS increased the strength of the dough, with significant increases in mixing time (MT) and peak width (PW), and decrease in breakdown in resistance (RBD) compared with the control. In addition, the dough with incorporation of the LMW-GS had more glutenin macropolyeric protein than the control, suggesting that the LMW-GS participated in forming larger glutenin polymers, and greatly contributed to dough strength. The changes in mixing parameters and the amount of glutenin macropolyeric protein were related to the quantity of incorporating subunits.

... 参考Xu等^[28]和Chen等^[29]的描述分析粉质参数 ...

2011

3.334

0.0

... 参考Xu等^[28]和Chen等^[29]的描述分析粉质参数 ...

1985

0.0

... 关于贮藏蛋白编码序列的内部出现终止密码的现象已有报道, 由密码子CAG (Gln)突变成TAG(终止密码)或由CAA (Gln)突变成TAA (终止密码)造成了1Ay、1Ax、1Dx不能正常表达, 成为了假基因, 由于碱基的转换突变引起的基因沉默同样也存在于醇溶蛋白基因中^{[30,31,32,33]} ...

2000

3.658

0.0

. 2000, 100:1085-1094 DOI:10.1007/s001220051390

Molecular characterisation of the inactive allele of the gene Glu-A1 and the development of a set of AS-PCR markers for HMW glutenins of wheat

1.Department of Cell Biology and Genetics, University of Alcalá, 28871 Alcalá de Henares, Madrid, Spain Tel.: +34-91 8854750, Fax +34-91 8854799 e-mail: bcjouve@gene.alcala.es ES

The present work reports new PCR markers that amplify the complete coding sequence of the specific alleles of the high molecular weight (HMW) glutenin genes. A set of AS-PCR molecular markers was designed which use primers from nucleotide sequences of the Glu-A1 and Glu-D1 genes, making use of the minor diffeences between the sequences of the x1, x2* of Glu-A1, and the x5 and y10 of Glu-D1. These primers were able to distinguish between x2* and the x1 or xNull of Glu-A1. Also x5 was distinguishable from x2, and y10 from y12. The primers amplified the complete coding regions and corresponded to the upstream and downstream flanking positions of Glu-A1 and Glu-D1. Primers designed to amplify the Glu-A1 gene amplified a single product when used with genomic DNA of common wheats and the xNull allele of this gene. This work also describes the cloning and characterisation of the nucleotide sequence of this allele. It possesses the same general structure as x2* and x1 (previously determined) and differs from these alleles in the extension of the coding sequence for a presumptive mature protein with only 384 residues. This is due to the presence of a stop codon (TAA) 1215-bp downstream from the start codon. A further stop codon (TAG), 2280-bp downstream from the starting codon is also found. The open reading frame of xNull and x1 alleles has the same size in bp. Both are larger than x2* which shows two small deletions. The reduced size of the presumptive mature protein encoded by xNull could explain the negative effect of this allele on grain quality.

2002

3.658

0.0

. 2002, 104:828-839 DOI:10.1007/s00122-001-0793-0

Isolation and characterization of five novel high molecular weight subunit of glutenin genes from Triticum timopheevi and Aegilops cylindrica

1.IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF, UK 2.The National Key Laboratory for Plant Cell and Chromosome Engineering, Institute of Genetics, The Chinese Academy of Sciences, Beijing 100101, China

1989

3.658

0.0

1993

3.658

0.0

... Zhong和Qualset^[34]通过提取种子全蛋白对122份来源不同的二倍体簇毛麦HMW-GS分析表明, 这些簇毛麦所编码的HMW-GS均在中国春1Dx2亚基和1Dy12亚基之间 ...

2004

0.599

0.0

. 2004, 46:489-496

High-molecular-weight glutenin subunit genes in decaploid Agropyron elongatum

通过SDS-PAGE法分析了高冰草(Agropyron elongatum(Host)Nevski)种子麦谷蛋白亚基,发现高冰草的麦谷蛋白亚基种类比普通小麦更加丰富.通过基因组PCR法用高分子量麦谷蛋白亚基基因的特异引物从高冰草核基因组中分离出了7条麦谷蛋白亚基的全编码序列,分别命名为AgeloGl～AgeloG7.其中的5条已进行全序列测定,对AgeloGl和AgeloG4进行了末端测序.尽管其中的4条基因的编码序列(AgeloG4,AgeloG5,AgeloG6和AgeloG7)小于1.8kb,但是对从克隆到的序列推导出的氨基酸序列与已经发表的小麦高分子量麦谷蛋白亚基序列进行对比分析发现,这些亚基与来自小麦的高分子量麦谷蛋白亚基具有很高的同源性.并且对信号肽、N-、C-末端的氨基酸序列分析显示,这7条序列编码的亚基皆为y-型亚基.用5条全部测序的编码序列与普通小麦的A、B、D、粗山羊草的D、圆柱山羊草的C、伞穗山羊草的U、黑麦的R染色体的编码高分子量麦谷蛋白的序列进行了聚类分析.表明,AgeloG2与小麦lDy,AgeloG3与小麦1By,AgeloG5、AgeloG6和AgeloG7与小麦1Ay在起源和进化上有较高的相似性.

Abstract：

Seven genes encoding glutenin subunits that present in Agropyron elongatum (Host) Nevski were cloned by PCR analysis and named AgeloG1 to AgeloG7. The complete open reading frames (ORFs) of the seven genes were amplified with primers special for high-molecular-weight (HMW) glutenin subunit genes and subsequently cloned and sequenced. Five of them were completely sequenced, and the other two (AgeloG1 and AgeloG4) were sequenced at the two ends only. Comparison of amino acid sequences suggested that the primary structure of the subunits encoded by the seven genes was very similar to that of Y-type HMW glutenin subunits published from wheat, though four of them (AgeloG4, AgeloG5, AgeloG6and AgeloG7) were shorter than 1.8 kb. Phylogenetic analysis of the five completely sequenced genes and those subunit genes of Triticum aestivum L. (AABBDD), Aegilops tauschii Coss. (DD), Aegilops caudata L.(CC), Secale cereale L. (RR) and Aegilops umbellulata Zhuk. (UU) indicated that the AgeloG2 was most closely related to 1 Dy; the AgeloG3 was to 1By; the AgeloG5, AgeloG6 and AgeloG7were to 1Ay.

... 8 kb的麦谷蛋白基因同样具有HMW-GS的特点^{[21,22,23,24,35,36,37,38,39,40,41,42]} ...

2004

0.0

1.321

. 2004, 24:237-242

Study on a novel HMW glutenin subunit coding region from Agropyron elongatum

(高冰草中一种新型高分子量谷蛋白亚基编码序列的研究) . (西北植物学报) , 2004 , 24 : 237 - 242 Feng D S , Chen F G , Zhao S Y , Xia G M . Study on a novel HMW glutenin subunit coding region from Agropyron elongatum

Feng D S

(封德顺) , (陈凡国) , (赵双宜) , (夏光敏) . (高冰草中一种新型高分子量谷蛋白亚基编码序列的研究) . (西北植物学报) , 2004 , 24 : 237 - 242 Feng D S

高冰草(Agropyron elongatun)是普通小麦(Triticum aestivum)的近缘禾草,SDS-PAGE显示其所编码的麦谷蛋白亚基的类型较普通小麦更加丰富,是普通小麦品质改良的重要亲本之一.利用基因组PCR的方法从高冰草中克隆到一个新的高分子量麦谷蛋白亚基(HMW-GS)基因(AgeloG2)全编码序列,同源性分析表明:与普通小麦的1Dy12基因比较在少数位点发生了碱基替换和一处6碱基序列的缺失,同源性为99%;与普通小麦的1Dy10基因比较,该基因亦只有少数碱基的替换和两处18碱基序列的增加及一处6碱基序列的缺失,同源性为98% .从推导的编码序列分析,AgeloG2编码y型HMW-GS.综上分析,AgeloG2是一个新的高分子量麦谷蛋白y-型亚基基因.聚类分析结果显示,无论在基因序列还是推导的氨基酸序列上,小麦1Dy亚基与AgeloG2的同源性都高于与粗山羊来源的y型亚基的同源性.

... 8 kb的麦谷蛋白基因同样具有HMW-GS的特点^{[21,22,23,24,35,36,37,38,39,40,41,42]} ...

2004

2.088

0.0

. 2004, 40:289-294 DOI:10.1016/j.jcs.2004.08.011

A novel high- molecular-weight glutenin subunit gene Ee1.5 from Elytrigia elongate (Host) Nevski

... 8 kb的麦谷蛋白基因同样具有HMW-GS的特点^{[21,22,23,24,35,36,37,38,39,40,41,42]} ...

2006

1.175

0.0

... 8 kb的麦谷蛋白基因同样具有HMW-GS的特点^{[21,22,23,24,35,36,37,38,39,40,41,42]} ...

2005

0.0

0.928

... 8 kb的麦谷蛋白基因同样具有HMW-GS的特点^{[21,22,23,24,35,36,37,38,39,40,41,42]} ...

2007

3.285

0.0

... 8 kb的麦谷蛋白基因同样具有HMW-GS的特点^{[21,22,23,24,35,36,37,38,39,40,41,42]} ...

2008

3.658

0.0

. 2008, 116:325-334 DOI:10.1007/s00122-007-0669-z

Characterizing HMW-GS alleles of decaploid Agropyron elongatum in relation to evolution and wheat breeding

1.Shandong University School of Life Sciences 250100 Jinan People’s Republic of China

Bread wheat quality is mainly correlated with high molecular weight glutenin subunits (HMW-GS) of endosperm. The number of HMW-GS alleles with good processing quality is limited in bread wheat cultivars, while there are plenty of HMW-GS alleles in wheat-related grasses to exploit. We report here on the cloning and characterization of HMW-GS alleles from the decaploid Agropyron elongatum. Eleven novel HMW-GS alleles were cloned from the grass. Of them, five are x-type and six y-type glutenin subunit genes. Three alleles Aex4, Aey7, and Aey9 showed high similarity with another three alleles from the diploid Lophopyrum elongatum, which provided direct evidence for the E^e genome origination of A. elongatum. It was noted that C-terminal regions of three alleles of the y-type genes Aey8, Aey9, and Aey10 showed more similarity with x-type genes than with other y-type genes. This demonstrates that there is a kind of intermediate state that appeared in the divergence between x- and y-type genes in the HMW-GS evolution. One x-type subunit, Aex4, with an additional cysteine residue, was speculated to be correlated with the good processing quality of wheat introgression lines. Aey4 was deduced to be a chimeric gene from the recombination between another two genes. How the HMW-GS genes of A. elongatum may contribute to the improvement of wheat processing quality are discussed.

... 8 kb的麦谷蛋白基因同样具有HMW-GS的特点^{[21,22,23,24,35,36,37,38,39,40,41,42]} ...

2010

0.0

0.928

... 8 kb的麦谷蛋白基因同样具有HMW-GS的特点^{[21,22,23,24,35,36,37,38,39,40,41,42]} ...