西农538 LMW-GS基因的克隆、原核表达及功能鉴定

为了探索普通小麦品种西农538的LMW-GS对面粉加工品质的影响,根据NCBI中已公布的LMW-GS基因序列,设计了一对特异性引物,从西农538基因组DNA中克隆出LMW-GS基因后,对其进行原核表达及掺粉试验。序列分析表明,克隆得到的LMW-GS基因序列(GenBank登录号为KX452081)有单一完整的开放阅读框,编码区长915bp,无内含子。同源性比对及进化树分析发现,该基因属于Glu-D3、Type V(Group 10)、m型、C组LMW-GS基因。SDS-PAGE和Western blot分析表明,该基因原核表达成功。微量掺粉试验表明,诱导表达的蛋白对小麦面粉加工品质有负效应。     

Polymerisation of gluten proteins in developing wheat grain as affected by desiccation

The breadmaking quality of wheat is affected by the composition of gluten proteins and the polymerisation of subunits that are synthesised and accumulated in developing wheat grain. The biological mechanisms and time course of these events during grain development are documented, but not widely confirmed. Therefore, the aim of this study was to monitor the accumulation of gluten protein subunits and the size distribution of protein aggregates during grain development. The effect of desiccation on the polymerisation of gluten proteins and the functional properties of gluten were also studied. The results showed that the size of glutenin polymers remained consistently low until yellow ripeness (YR), while it increased during grain desiccation after YR. Hence, this polymerisation process was presumed to be initiated by desiccation. A similar polymerisation event was also observed when premature grains were dried artificially. The composition of gluten proteins, the ratios of glutenin to gliadin and high molecular weight-glutenin subunits to low molecular weight-glutenin subunits, in premature grain after artificial desiccation showed close association with the size of glutenin polymers in artificially dried grain. Functional properties of gluten in these samples were also associated with polymer size after artificial desiccation.     

Kernel vitreousness and protein content: Relationship,interaction and synergistic effects on durum wheat quality

Four sets of durum samples were used in this study to further understand the interrelationships among hard vitreous kernels (HVK), protein content, and pigment concentration, with a focus on the interaction and synergistic effects of protein content and vitreousness on durum quality. HVK level increases with higher protein content in the range of 9.5–12.5%, but this relationship is less evident in durum samples with high protein content (12.5–14.5%). Both protein content and kernel vitreousness can significantly affect durum milling quality. White starchy kernels (WSK) in low protein durum have a very detrimental impact on milling and pasta processing quality, but high protein content can mitigate the adverse impact of WSK on durum quality. Although protein content plays a dominant role, higher HVK might contribute positively to pasta firmness. There was no significant difference in yellow pigment content between HVK and WSK. However, pigment loss from semolina to dough was higher for WSK than HVK. Despite the difference in protein content, HVK and WSK have little difference in gluten strength. The monomeric protein was preferentially accumulated in HVK. The glutenin proteins of HVK and WSK were similar in the ratios of 1Bx/1By and HMW/LMW-GS.     

冬播麦区Glu-1和Glu-3位点变异及1B/1R易位与小麦加工品质性状的关系

 贮藏蛋白组成是决定小麦加工品质的重要因素。本文调查了我国冬播麦区251份主栽品种和高代品系的高分子量麦谷蛋白亚基（HMW-GS）、低分子量麦谷蛋白亚基（LMW-GS）和1B/1R易位的分布状况，研究了它们与加工品质性状的关系。结果表明，品质较差的HMW-GS N、7+9、2+12和LMW-GS Glu-A3a与Glu-B3j（1B/1R易位）在冬播麦区分布较广，频率分别为39.4%、45.0%、59.8%、37.1%和44.6%。HMW-GS和LMW-GS等位变异对籽粒蛋白质含量影响较小，对SDS沉降值、和面时间与耐揉性的加性和互作效应达1%的显著水平。按位点对加工品质性状的贡献大小，Glu-D1>Glu-B3>Glu-B1>Glu-A3>Glu-A1；就单个亚基而言，Glu-A1位点，1>2*>N；Glu-B1位点，7+8>14+15>7+9；Glu-D1位点，5+10>4+12>2+12；Glu-A3位点，Glu-A3d>Glu-A3a>Glu-A3c>Glu-A3e，Glu-B3位点； Glu-B3d>Glu-B3b>Glu-B3f >Glu-B3j。1B/1R易位对SDS沉降值、和面时间和耐揉性等加工品质性状有显著负面效应。通过选择优质高低分子量麦谷蛋白亚基和淘汰1B/1R易位系，将有助于提高我国小麦的面筋质量。     

钩刺山羊草(Aegilops triuncialis)低分子量谷蛋白基因序列分析

根据低分子量谷蛋白亚基(LMW-GS)基因编码区保守序列设计引物,对钩刺山羊草(AegilopstriuncialisL,2n=4x=28,CCUU)PI483029总DNA进行PCR扩增,得到长度为900和1065bp的DNA片段,克隆测序后获得2个LMW-GS基因,GenBank登录号分别为AY841016和AY841017。它们具有小麦LMW-GS基因的典型结构特征。其中,AY841017具有完整编码区,长度为1065bp,可编码322个氨基酸残基的成熟蛋白,第一个半胱氨酸残基出现在重复区第13位。在重复区,AY841017的两个疏水单元为PIIIL和PVIIL,重复区中还存在一个连续13个Q(谷氨酰胺)组成的短肽。AY841016由于编码区内存在提前终止密码子,为假基因。氨基酸序列比较发现,AY841017与普通小麦(IriticumaestivumL.)Glu-D3位点编码的LMW-GS基因有很高的一致性。     

Evaluation of fresh pasta-making properties of extra-strong common wheat (Triticum aestivum L.)

The relationship between characterictics of flour of common wheat varieties and fresh pasta-making qualitites was examined, and the fresh pasta-making properties of extra-strong varieties that have extra-strong dough were evaluated. There was a positive correlation between mixing time (PT) and hardness of boiled pasta, indicating that the hardness of boiled pasta was affected by dough properties. Boiled pasta made from extra-strong varieties, Yumechikara, Hokkai 262 and Hokkai 259, was harder than that from other varieties and commercial flour. There was a negative correlation between flour protein content and brightness of boiled pasta. The colors of boiled pasta made from Yumechikara and Hokkai 262 grown under the condition of standard manuring culture were superior to those of boiled pasta made from other varieties. Discoloration of boiled pasta made from Yumechikara grown under the condition of heavy manuring culture was caused by increase of flour protein content. On the other hand, discoloration of boiled pasta made from Hokkai 262 grown under the condition of heavy manuring culture was less than that of boiled pasta made from Yumechikara. These results indicate that pasta made from extra-strong wheat varieties has good hardness and that Hokkai 262 has extraordinary fresh pasta-making properties.     

小麦Glu-3位点基因拷贝数的变异分析

【目的】基因拷贝数变异是一种常见又重要的基因结构变异,往往影响个体表型。低分子量麦谷蛋白（low-molecular-weight glutenin subunit,LMW-GS）是小麦贮藏蛋白的主要组成部分,位于Glu-3位点。小麦作为异源六倍体,其庞大且复杂的基因组结构导致难以利用传统方法检测目的基因的拷贝数,针对小麦基因组,筛选可靠稳定的内参基因和体系,探索适合复杂基因组的拷贝数变异测定技术,测定Glu-3位点LWM-GS基因拷贝数。【方法】以Acc1为内参基因,根据基因序列设计内参引物和探针,通过定性和定量PCR测定内参基因在12个普通小麦品种中的拷贝数,分析该基因拷贝数在不同品种间的稳定性;又以小麦品种篙优2018的5个稀释浓度的基因组DNA为模板,利用qRT-PCR验证Acc1内参系统的重复性和准确性;根据Glu-A3位点LMW-GS基因序列设计特异性引物及探针,利用qRT-PCR和ddPCR 2种方法检测8个小麦品种Glu-A3位点基因拷贝数,比较后选择更优的高通量基因拷贝数检测方法;再根据Glu-B3和Glu-D3位点LMW-GS基因序列设计相应的特异性引物及探针,并利用ddPCR技术检测和分析了231份小麦品种的Glu-A3、Glu-B3和Glu-D3位点上LMW-GS基因拷贝数。【结果】Acc1在12个普通小麦品种间、同一品种5个DNA稀释浓度间的拷贝数测定结果一致,技术重复间的变异系数仅为0.07%—0.77%,所构建的Acc1内参系统稳定;比较qRT-PCR和ddPCR 2种拷贝数检测方法,8个品种所测的Glu-A3位点拷贝数结果一致,分别为3、5、3、4、3、3、3和3;且ddPCR检测重复间的变异系数为0.30%—1.67%,远低于qRT-PCR的3.14%—12.72%,更加可靠;利用ddPCR对231份普通小麦品种的Glu-A3、Glu-B3和Glu-D3位点上LMW-GS基因拷贝检测后分析发现,大多数小麦品种在3个位点上的拷贝数为4,所占频率分别为51.95%、32.03%和28.57%,Glu-3位点总拷贝数变异范围为10—21,变异系数为16.12%。【结论】Acc1内参系统具有良好的稳定性和重复性,可以用作小麦Glu-3位点和其他目的基因拷贝数检测的内参;qRT-PCR和ddPCR均可用于小麦基因拷贝数的检测,但后者更稳定、可靠,且操作简单、检测通量高。     

Metabolite profiling of the response to high-nitrogen fertilizer during grain development of bread wheat (Triticum aestivum L.)

Wheat yield and quality are dependent largely on nitrogen (N) availability. In this study, we performed the first metabolomic analysis of the response to high-N fertilizer during wheat grain development using non-targeted gas chromatography-mass spectrometry (GC–MS). Quality parameter analyses demonstrated that high-N fertilizer application led to a significant increase in grain protein content and improvement in starch and bread-making quality. Comparative metabolomic profiling of six grain developmental stages resulted in identification of 74 metabolites, including amino acids, carbohydrates, organic acids and lipids/alcohol, which are primarily involved in carbon and N metabolism. Under high-N fertilizer treatment, numerous metabolites accumulated significantly during grain development. Principal component analysis revealed two principal components as being responsible for the variances resulting from N-fertilizer treatments. Metabolite–metabolite correlation analysis demonstrated that the high-N treatment group had a greater number of positive correlations among metabolites, suggesting that high-N fertilizer treatment induced a concerted metabolic change that resulted in improved grain development. Particularly, the high-N treatment-mediated significant accumulation of metabolites involved in the TCA cycle, starch and storage protein synthesis could be responsible for the improvement of grain yield and quality. Our results provide new insight into the molecular mechanisms of wheat grain development and yield and quality.     

宁夏小麦Glu-A3与Glu-B3位点等位变异组成分析

低分子量谷蛋白亚基(LMW-GS)与小麦品质密切相关。为给宁夏小麦的品质改良提供参考,应用STS分子标记,对宁夏98份小麦品种Glu-A3和Glu-B3位点的等位基因变异类型组成进行检测和分析。结果表明,宁夏98份小麦品种Glu-A3位点存在Glu-A3a(4.0%)、Glu-A3b(2.0%)、Glu-A3c(55.1%)、GluA3d(10.1%)和Glu-A3e(28.5%)5种等位变异;Glu-B3位点存在Glu-B3a(2.0%)、Glu-B3b(8.1%)、Glu-B3c(5.0%)、Glu-B3d(7.1%)、Glu-B3f(21.4%)、Glu-B3g(2.0%)、Glu-B3h(15.3%)和Glu-B3i(13.3%)8种等位变异。宁夏参试品种共存在23种等位变异组合形式,其中引黄灌区存在18种组合类型,Glu-A3c/Glu-B3j(17.7%)和Glu-A3e/Glu-B3f(16.2%)组合类型较为普遍;宁南山区存在13种组合类型,以Glu-A3c/Glu-B3i(20.0%)组合类型为主。在参试的宁夏小麦品种中,对同一地区不同来源品种而言,改良品种的LMW-GS遗传多样性明显较引进品种和地方品种更丰富。     

Influence of allelic prolamin variation and localities on durum wheat quality

Thirty-seven varieties of a Mediterranean durum wheat collection grown in Tunisia and Spain were analysed for their allelic composition in prolamins, as well as their protein concentration, sodium dodecyl sulphate sedimentation (SDSS) test and mixograph parameters. Genotype was a greater source of variation in all measurements than locality. Uncommon high and low molecular glutenin subunits (HMW-GS and LMW-GS) were found (V and 2•• subunits at Glu-A1, 13 + 16 at Glu-B1, 5* subunit and ax allele at Glu-A3). The rare combinations 2 + 4+14 + 18 and 8 + 9+13 + 16+18 subunits at the Glu-B3 locus were found. Glu-A3ax had a positive influence on SDSS and mixograph parameters. Of all the prolamins, those that have the B-LMW-GS composition aaa (for Glu-A3, Glu-B3 and Glu-B2 loci, respectively), when associated with the Glu-A1c and Glu-B1d gave the best semolina quality. By contrast, semolina quality is poor when this same composition is associated with the Glu-A1c and Glu-B1e and even poorer when associated with the Glu-A1c and Glu-B1f. In addition, the cultivars with B-LMW-GS allelic composition aab (for Glu-A3, Glu-B3 and Glu-B2 loci, respectively), when associated with the Glu-A1c and Glu-B1d, gave high quality, whereas when associated with the Glu-A1c and Glu-B1e or with Glu-A1o and Glu-B1f, the quality was very poor.