HMW-GS分子标记多重PCR体系的建立及新疆春小麦的检测

为给新疆优质春小麦品种选育提供参考依据,利用小麦优质高分子量谷蛋白亚基(HMW-GS)基因的特异性标记,基于17份已知HMW-GS组成的品种(系),构建了3套多重PCR,体系Ⅰ可用于同时检测AxNull和Dx5基因,体系Ⅱ可同时检测Ax2*和By8基因,体系Ⅲ可同时检测Bx14和Dx5基因;用3套多重PCR体系分别检测17份小麦品种,其结果与SDS-PAGE检测结果完全一致,表明建立的3套多重PCR体系稳定可靠,可用于小麦品种优质HMW-GS基因聚合育种.利用此体系对85份新疆春小麦品种进行分析表明,AxNull和Ax1的频率均为24.7%,Ax2*为50.6%,By8为48.2%,Bx14(+By15)为0%,Dx5(+Dy10)为34.1%.     

小麦高分子量谷蛋白亚基基因分子育种研究进展

为给小麦品质改良工作者提供通过分子生物技术优化HMW-GS组成方面的全面信息,综述了HMW-GS的基因克隆、分子标记以及基因工程改良三个方面近年来的国内外研究进展.迄今为止, 被克隆和测序的HMW-GS基因已有20多个,即1Ax1、1Ax2*、1Ax2*B、1Ay1、1Bx7、1Bx9、1Bx17、1Dx2、1Dx5、1Bx20、1By8、1By9、1Dy10、1Dy12、1AxNull、1Bx14、 1Bx23、1Dx2.2、1Dx2.1、1Dy10.1、1Dy12t等,还不断有新的基因被发现和克隆.克隆方法可概括为两种:一种是以HMW-GS克隆作为探针筛选cDNA或基因组DNA文库, 从而获得所需的靶基因序列, 然后再选择合适的载体进行克隆测序;另一种则是采用PCR技术.HMW-GS基因的分子标记方法主要有RFLP法、PCR法和SNP(单核苷酸多态性)法.目前已有研究者通过基因工程方法将部分外源HMW-GS基因导入小麦,有效地改善了受体品种的加工品质.     

小麦体细胞杂种渐渗系F6代高分子量谷蛋白亚基组成与位点变异研究

为了发掘新的小麦高分子量谷蛋白用于面粉优质育种工作,本文测定了小麦/长穗偃麦草体细胞杂种共322个株系的高分子量麦谷蛋白亚基（HMW-GS）组成,并根据Payne等人1987年制定的小麦品质评分标准进行了品质评分。结果表明,体细胞杂种株系在Glu–A1,Glu–B1和Glu–D1位点上的遗传变异率分别为0.52,0.58和0.46,一共出现了27种不同的亚基组成形式,其中H1AxNull,H1Bx7+H1By9,H1Dx2+H1Dy12（同亲本小麦济南177）出现的频率最高,而H1Ax2*,H1Bx7+H1By8+H1By9,H1Dx2+H1Dx5+H1Dy12出现的频率最低;H1Dx5+H1Dy12出现的频率约为33.5%;在与优质可能相关的亚基组合当中,H1Bx13+H1By16出现的频率约为31.1%。由于1Dx5+1Dy12尚未有品质评分,因此,部分株系无法给出分数。该实验结果表明通过体细胞杂交可以产生大量的高分子量谷蛋白亚基/组合变异,这将有助于今后小麦面粉品质育种工作。     

离子束诱导小偃81突变系麦谷蛋白和醇溶蛋白遗传变异分析

摘要：本实验以N+（30Kev）注入诱导获得的小偃81突变系M4代种子为材料,采用SDS-PAGE和A-PAGE技术对其高分子量麦谷蛋白亚基和醇溶蛋白进行系统分析,检测到3个高分子量麦谷蛋白亚基缺失系：1Ax1缺失系,1Bx14+1By15缺失系,1Dx2+1Dy12缺失系,出现频率由大到小依次为1Ax1＞1Bx14+1By15＞1Dx2+1Dy12。醇溶蛋白方面共检测到5种变异类型,1Ax1缺失系有3种突变类型,1Bx14+1By15缺失系和1Dx2+1Dy12缺失系各有1种突变类型,ω区变异类型最多,其次是α区和γ区,β区没有发现变异类型,在5种变异类型中有一条相同的变异谱带。结果表明：N+（30Kev）离子束注入能有效地诱导小麦种子高分子量谷蛋白亚基和醇溶蛋白的变异,并能够在后代中稳定遗传。     

面包面条兼用型强筋小麦主要品质性状分析与评价

为明确面包面条兼用型强筋小麦主要品质水平,对32份集聚Ax2^*/Bx7+By8或By9/Dx5+Dy10亚基和 wx-B1b基因的春小麦品种(系)进行面粉品质相关指标分析。结果表明,供试品种(系)面筋指数平均为94.4%,能量平均为122 cm²,延伸性平均为146 mm,最大拉伸阻力平均为654 EU;糊化温度平均为63.7℃,峰值粘度平均为1 125 BU,峰值温度平均为87.4℃,各品质指标表现优良。对近年审定的4个品种进行面制品评分发现,面包评分平均为84.3,面条评分平均为86.2,饺子皮评分平均为84.3,均属于优质面包面条兼用型品种。对品质指标进行相关性分析发现,面筋指数、拉伸参数等与淀粉糊化特性相关不显著,面筋质量与淀粉特性可实现同步改良。Ax2^*/Bx7+By8或By9/Dx5+Dy10/ wx-B1b可作为兼用型强筋小麦优质基因集聚类型之一,在品质改良中加以利用。本研究中,龙19-9859、龙19-9876、龙19-9878、龙18H2305、龙蒙麦9030、龙麦77、龙麦86及龙麦94为优异面包面条...     

小麦HMW GS检测方法的优化及应用

为了准确快速鉴定小麦品种的HMW-GS组成,以18个已知HMW-GS组成的品种为对照,将4种不同浓度分离胶的SDS-PAGE与分子标记(Bx7、Bx7OE和By 8)相结合,构建一套适于检测HMW-GS组成的方法,并用该方法检测214份陕西小麦品种(系)的HMW-GS组成。4种分离胶浓度中,除了亚基7、7*与7OE和8与8*外,9%的分离胶可以很清楚地分离Glu1-位点的其他常见亚基,结合分子标记(Bx7、Bx7OE和By 8)检测对照品种,结果与已知亚基(基因)一致。用分离胶浓度为9%的SDS-PAGE结合分子标记(Bx7、Bx7OE和By 8)方法对陕西品种(系)检测结果表明,Glu-A1、Glu-B1和Glu-D1分别有3、8和3个亚基种类,共28种亚基组合类型。以1、Null、7+9、7+8、7+8*、14+15、2+12和5+10为主要亚基类型,频率分别为55.6%、41.6%、43.9%、26.2%、10.3%、15.4%、79.0%和12.6%;同时在Glu-B1位点发现7+8*和6+8*亚基。该方法可以快速准确地评价小麦HMW-GS组成,有效区分Glu-B1位点的亚基7与7OE,8与8*,鉴定结果稳定可靠。     

甘肃冬小麦品种(系)面粉品质性状相关基因分析

为了解甘肃省近20年育成的106份冬小麦品种(系)中加工品质性状相关基因分布情况,用22个分子标记对供试材料的HMW-GS、LMW-GS、面粉色泽及籽粒硬度等品质性状相关基因进行了分析。结果发现,供试品种(系)的HMW-GS相关基因中,在Glu-A1位点检测到34份品种(系)含有AxNull,频率为32.08%;在Glu-B1位点检测到Bx7+By8和Bx14+By15共2种基因组合,分别占17.92%和25.47%;在Glu-D1位点检测到11份品种(系)含有Dx5+Dy10,占10.38%。对LMW-GS鉴定结果显示,29份品种(系)含Glu-A3d基因,分布频率为27.36%。HMW-GS和LMW-GS亚基组合中,含有4个、3个和2个位点优质亚基基因组合的品种(系)分别占0.94%、8.49%和3.77%。对面粉色泽相关基因Ppo-A1、Ppo-D1、Psy-A1、Lox-B1和TaPod-A1位点的检测发现,优异等位变异占比分别为39.62%、50.94%、31.13%、30.19%和38.68%。对籽粒硬度相关基因检测发现,在Pina、Pinb和Pinb-2等位变异位点的检测...     

HMW-GS分子标记多重PCR体系的建立及新疆春小麦的检测

为给新疆优质春小麦品种选育提供参考依据,利用小麦优质高分子量谷蛋白亚基(HMW-GS)基因的特异性标记,基于17份已知HMW-GS组成的品种(系),构建了3套多重PCR,体系Ⅰ可用于同时检测AxNull和Dx5基因,体系Ⅱ可同时检测Ax2*和By8基因,体系Ⅲ可同时检测Bx14和Dx5基因;用3套多重PCR体系分别检测...     

黄淮南片小麦高分子量谷蛋白亚基组成及其与品质的关系

为了解小麦高分子量谷蛋白亚基(HMW-GS)组成与品质之间的关系,对177份黄淮南片地区小麦品种(系)的HMW-GS组成及其品质进行了检测。结果表明,177份材料在Glu-A1位点上有3种亚基类型(N、1、2*),在Glu-B1位点上有5种亚基类型(7+8、7+9、14+15、17+18、13+16),在Glu-D1位点上有3种亚基类型(2+12、5+10、5+12),1、14+15、5+10亚基的出现频率分别为73.45%、11.86%和48.02%。1、7+8、17+18、5+10亚基对蛋白质含量、湿面筋含量、峰值曲线面积、峰高、SDS沉淀值、8min带高、8min曲线面积、形成时间、稳定时间都具有较大的正向效应。共检测到19种不同的亚基组合类型,其中1/7+8/5+10亚基组合的小麦品种(系)各项品质指标均较优,其次是1/7+8/2+12亚基组合品种(系),N/7+9/2+12和N/14+15/5+10亚基组合类型小麦的品质较差。     

高效毛细管电泳（HPCE）对优质小麦HMW-GS的分离鉴定

为了解应用高效毛细管电泳(HPCE)技术定量分析优质小麦高分子量谷蛋白亚基(HMW-GS)的效果,利用HPCE技术对13个小麦品种的HMW-GS进行亚基识别和定量分析,并与传统的SDS-PAGE电泳图进行了比较.结果表明,在HPCE电泳图中HMW-GS的亚基出现顺序与SDS-PAGE略有不同,亚基1Ax1早于1Bx7出峰,并且在主亚基之后还有小的亚基出现,尤其是1Dx2主亚基后面有3个小亚基.定量分析表明,优质小麦品种陕627的HMW-GS,不论单个亚基、亚基组合还是亚基总和的含量都高于其他小麦品种,且均达极显著水平.优质小麦品种陕715的HMW-GS含量除亚基1Ax1与陕627接近外,其余亚基和亚基组合都显著低于陕627.西农5211的HMW-GS因为不舍有亚基1Ax1,尽管其他亚基、亚基组合含量接近于陕627,但HMW-GS的总含量显著低于陕627.优质小麦的优质亚基含量较高.HPCE具有高效、灵敏、再现性高等特点,能够快速识别和定量分析小麦HMW-GS,可以为小麦品质育种或组合选择提供参考.     

57份陕西新育成小麦品种（系）HMW-GS组成及品质分析

为探究陕西关中地区小麦HMW-GS亚基与品质性状间的关系,采用SDS-PAGE法对57份陕西关中地区小麦品种（系）HMW-GS亚基组成及相关品质性状进行了分析。结果表明,供试品种（系）中共检测出7种HMW-GS亚基类型和8种HMW-GS亚基组合;Glu-A1位点上有3种亚基类型,分别为1、2^*和Null,以1亚基为主（78.95%）;Glu-B1位点上检测到7+8（61.40%）与7+9（38.60%）两个类型;Glu-D1位点上检测到5+10（70.18%）和2+12（29.82%）两个类型。3个HMW-GS基因位点编码亚基共组成8种亚基组合,品质得分6～10分,其中1/7+8/5+10组合品质得分10分,出现频率最高。就HMW-GS不同位点对品质性状效应进行分析发现,Glu-D1位点对b^*值、形成时间、稳定时间、弱化度和粉质质量指数的影响达到极显著水平（P＜0.01）;对面团流变学特性的影响,Glu-D1>Glu-B1。不同类型亚基对小麦品质的效应存在差异,7+8亚基对蛋白质含量、湿面筋含量和容重具有正效应,7+9和5+10亚基对形成时间和稳定时间的影响显著高于其他亚基（P＜0.05）;携带1/7+8/5+10亚基组合小麦的蛋白质、湿面筋含量和容重最高;携带1/7+9/5+10亚基组合具有较高面粉L^*值和面团流变学特性指标值。     

Stacking HMW-GS transgenes in bread wheat: Combining subunit 1Dy10 gives improved mixing properties and dough functionality

We have determined the technological properties of four lines containing combinations of three HMW-GS transgenes, encoding HMW-GS 1Ax1, 1Dx5 and 1Dy10. These lines were produced by conventional crossing of three single transgenic lines of the bread wheat cultivar Anza that contains the endogenous HMW-GS pairs 1Dx2 + 1Dy12 and 1Bx7* + 1By8 and is null for the Glu-A1 locus. Consequently, the total number of HMW-GS ranged from 4 in the control line Anza to 7 in line T618 which contains all three HMW-GS transgenes. The lines were studied over two years using a range of widely used grain and dough testing methods. All lines with transgenic subunits showed higher levels of glutenin proteins than the Anza control, and these differences were highly significant for lines T616, T617 and T618, containing, respectively, the transgenes encoding HMW-GS 1Ax1 and 1Dy10, 1Dx5 and 1Dy10 and 1Ax1, 1Dx5 and 1Dy10. These increases in glutenin levels are compensated by lower levels of gliadins present in transgenic lines. These changes affected the ratio of polymeric to monomeric gluten proteins (poly:mono), the ratio of HMW-GS to LMW-GS (HMW:LMW) and the contents of individual 1Ax, 1Bx, 1By, 1Dx and 1Dy subunits. Transgenic lines expressing subunit 1Dy10 together with x-type subunits (T616, T617 and T618) were superior to line T606, which had only increases in x-type subunits. In particular, the combination of transgenic subunits 1Dx5 and 1Dy10 (line T617) gave better dough rheological properties than the other combinations of transgenic subunits. For example, dough development time and stability were increased by 3.5-fold and 8.5-fold, respectively, while the mixing tolerance index (MTI) was decreased by 3.3-fold in line T617 with respect to the control line. Alveograph analyses showed that all four transgenic combinations had increased P values compared to the Anza control but subunit 1Dx5 greatly reduced the extensibility (L). These results show that stacking HMW-GS transgenes by conventional crossing is a valid strategy for the improvement of wheat quality, with different effects being related to the different HMW-GS combinations.     

Mixing properties and dough functionality of transgenic lines of a commercial wheat cultivar expressing the 1Ax1, 1Dx5 and 1Dy10 HMW glutenin subunit genes

In this work we report the effects of the HMW-GS 1Ax1, 1Dx5 and 1Dy10 on the breadmaking quality of the bread wheat cultivar Anza that contains the HMW-GS pairs 1Dx2 + 1Dy12 and 1Bx7* + 1By8, and is null for the Glu-A1 locus. This allows the characterization of individual subunits 1Dx5 and 1Dy10 in the absence of subunit 1Dx5, and the interactions between these subunits and subunits 1Dx2 and 1Dy12 to be determined. Three transgenic lines termed T580, T581 and T590, containing, respectively, the HMW-GS 1Ax1, 1Dx5 and 1Dy10 were characterized over 3 years using a range of widely-used grain and dough testing methods. The transgenic subunits 1Ax1, 1Dx5 and 1Dy10 accounted for 25.2%, 20.3% and 17.9%, respectively, of the total HMW-GS in the three transgenic lines. Although lines T581 and T590 expressed similar levels of subunits 1Dx5 and 1Dy10 they had different effects on other aspects of protein composition, including changes in the ratios of glutenin/gliadin, of HMW/LMW-GS, the 1Dx2/1Dy12, the x-type/y-type HMW-GS and the proportions of high molecular mass glutenin polymers. In contrast, lines transformed to express subunits 1Ax1 and 1Dx5 showed similar changes in protein composition, with higher protein contents and decreased ratios of glutenin/gliadin and 1Dx2/1Dy12. In addition, both transgenic lines showed similar increases in the ratio of x-type/y-type subunits compared to the control line. The transgenic lines were analysed using Farinograph, Mixograph and Alveograph. This confirmed that the expression of all three subunits resulted in increased dough strength (and hence breadmaking quality) of the cultivar Anza. A beneficial effect of subunit 1Dx5 has not been reported previously, transgenic wheat lines expressing this subunit giving overstrong dough unsuitable for breadmaking. However, the expression of subunit 1Dy10 had a greater effect on breadmaking quality than subunits 1Ax1 and 1Dx5. The Farinograph parameters such as dough stability and peak time were increased by 9.2-fold and 2.4-fold, respectively, in line T590 (expressing 1Dy10) with respect to the control line. Similarly, the Mixograph mixing time was increased by four-fold and the resistance breakdown decreased by two-fold in line T590 compared with the control line. The Alveograph W value was also increased by 2.7-fold in line T590 compared to the control line. These transgenic lines are of value for studying the contribution of specific HMW-GS to wheat flour functional properties.     

氮肥运筹对小麦HMW GS形成与积累的影响

为给小麦优质栽培提供理论依据,以多穗型品种031085和大穗型品种山农91为试验材料,利用SDSPAGE电泳等分析技术,研究了氮肥运筹对不同类型小麦品种高分子量谷蛋白亚基（HMWGS）形成的影响。结果表明,小麦HMWGS出现的时间多在花后10~15 d,且多穗品种相对早于大穗品种;氮肥运筹对HMWGS出现时间影响不大;亚基组合1Bx7+1By9出现时间早于亚基1Dx2+1Dy12;不同施氮处理下,开花后各小麦品种HMWGS亚基的积累过程均呈三次曲线增长;各亚基积累量所占百分含量随时间变化较为平稳,受施氮影响较小,y型亚基百分含量高于x型亚基。     

SDS-PAGE与分子标记相结合分析宁夏小麦HMW-GS组成与变化特点

为了建立准确有效小麦HMW-GS的检测方法,提高优质小麦品种鉴定和筛选效率,以已知HMW-GS组成的16份小麦品种为对照,优化完善SDS-PAGE结合分子标记检测小麦HMW-GS的方法,并对103份宁夏小麦品种进行了验证和分析。结果表明,8.5%的分离胶可以有效区分除了 Glu-B1位点的7^*、7^OE与8^*亚基之外的其他亚基,分辨效果优良;SDS-PAGE结合 Bx7^OE、 Bx7^*/Bx7和 By8基因的分子标记可以准确鉴定小麦HMW-GS。在103份宁夏小麦品种中,发现15种HMW-GS和29种组合类型;首次在该地区小麦品种的 Glu-B1位点检测出了携带7^OE、7^*和8^*亚基的品种;1/17+18/5+10为当地小麦HMW-GS的优势亚基组合类型,占20%。自1970年至2010年,HMW-GS的优质亚基（1、17+18和5+10）的出现频率呈明显增长趋势;宁夏小麦的HMW-GS种类和优质亚基出现频率随品种更换呈增加趋势。综上所述,分离胶浓度为8.5% 的SDS-PAGE和 Bx7^OE、 Bx7^*/Bx7和 By8分子标记的方法可准确有效地检测小麦HMW-GS组成,此方法可用于优质小麦品种的鉴定和筛选。     

Pasting properties of transgenic lines of a commercial bread wheat expressing combinations of HMW glutenin subunit genes

Seven transgenic lines of a commercial wheat (Triticum aestivum L.) cultivar expressing transgenic subunits 1Ax1, 1Dx5 and 1Dy10, alone or in combination have been developed. Pasting properties were determined in these transgenic lines using a Rapid Visco Analyser (RVA) in order to determine the possible impact of HMW-GS transgene expression on the starch properties. Expression of the HMW-GS transgenes increased the proportions of the corresponding 1Ax, 1Dx and 1Dy subunits affecting significantly the ratios of HMW-GS:LMW-GS and x-type:y-type HMW-GS. Starch granule size distribution varied significantly among all transgenic lines, with the Anza control and transgenic line T616 (expressing subunits 1Ax1 and 1Dy10) showing the highest and the lowest percentage of B granules, respectively. All transgenic lines increased the water-binding capacities (WBC) at 25 °C and 90 °C. Line T606 (expressing subunits 1Ax1 and 1Dx5) and line T590 (expressing subunit 1Dy10) showed the lowest and the highest values for peak viscosity, respectively. Notably, lines expressing only transgenic x-type subunits (T580, T581 and T606), with high ratios of x-type:y-type HMW-GS, had low peak viscosities, final viscosities and breakdown viscosities. Line T590 had the highest breakdown viscosity while lines T606 and T581 had the lowest.     

Molar fractions of high-molecular-weight glutenin subunits are stable when wheat is grown under various mineral nutrition and temperature regimens

Molar fractions of the high-molecular-weight glutenin subunits (HMW-GS) were determined for flour from bread wheat (Triticum aestivum L. cv Butte86) produced under 13 different combinations of temperature, water and mineral nutrition. Albumins, globulins and gliadins were removed from the flour by extraction with 0.3 M NaI in 7.5% 1-propanol. Total HMW-GS were recovered by extracting the remaining protein with 2% SDS and 25 mM DTT. Individual HMW-GS were then separated and quantified by RP-HPLC. Constant molar fractions for the five HMW-GS were maintained under all environmental conditions, despite large differences in duration of grain fill, total protein per grain, flour protein percentage, and total HMW-GS per grain. Similar molar fractions were found for five other US wheat varieties. The Bx7 subunit accumulated to the highest level at 30% of total HMW-GS. The Dx and Dy subunits were present in smaller but nearly equal proportions, 22% and 23%, respectively, and the Ax and By subunits were the least abundant, 14% and 12%, respectively. Although the amounts of HMW-GS per unit of flour are strongly affected by environment, the different subunits respond so similarly to external conditions that their final proportions appear to be determined mainly by genetic factors.