条锈菌诱导上调表达的小麦腺苷甲硫氨酸脱羧酶（SAMDC）基因的克隆及其特征分析

为了克隆条锈菌诱导上调表达的小麦腺苷甲硫氨酸脱羧酶（SAMDC）基因并研究其在小麦感病、抗病单株苗期抗条锈病防御反应中的作用,以大麦（Hordeum vulgareL.）SAMDC全长cDNA序列为信息探针,采用电子克隆、RACE（Rapid amplification of cDNA ends）和RT-PCR方法,从条锈菌（Puccinia stri-iformisf.sp.tritici）CYR32侵染的小麦（Triticum aestivumL.）抗条锈病新种质NR1121中分离出1个新的小麦SAMDC基因家族成员,命名为TaSAMDC2（GU016570）。TaSAMDC2基因cDNA序列全长2 003 bp,5′非翻译区区域和一个带有Poly（A）的3′非翻译区区域长分别为553和283 bp;该基因的开放阅读框为1 167 bp,编码388个氨基酸,编码的氨基酸序列包含酶原剪切位点和PEST结构域。基因组序列全长2 539bp,位于5′UTR存在一个526 bp长的内含子序列,内含子的剪切位点均符合真核生物GT-AG规则。同源序列分析表明,TaSAMDC2与来自大麦、水稻（Oryza sativaL.）、玉米（Zea maysL.）、一粒小麦（TriticummonococcumL.）4种植物SAMDC蛋白的相似性分别为95.0%、85.0%、80.0%和80.0%。半定量RT-PCR与实时荧光定量PCR分析表明,TaSAMDC2的表达受条锈菌诱导,小麦苗期经条锈菌侵染后,在抗病材料中,该基因于48 hpi上调表达至最高水平,而在感病材料中先下调、上调表达至最高水平明显滞后。结果提示,分离到的是一个条锈菌CYR32诱导后上调表达的小麦SAMDC基因,该基因可能参与了小麦的抗条锈病反应。     

橡胶树肌动蛋白解聚因子原核表达及纯化

本研究根据HbADF序列设计引物扩增基因的编码区,并将其插入到原核表达载体pET28a上,成功构建重组质粒pET28a-HbADF。将重组质粒转化大肠杆菌BL21(DE3)宿主菌,经1 mmol/L IPTG诱导,获得相对分子量为20 ku的融合蛋白。表达蛋白以可溶和包涵体两种形式存在,通过亲和层析方法纯化可溶蛋白并获得HbADF融合蛋白,用抗HIS标签的单抗对纯化蛋白进行了Western blot鉴定。该结果为进一步研究HbADF蛋白特性及功能奠定基础。     

小麦冷休克蛋白基因TaCSP的克隆及表达分析

为深入研究小麦冷休克蛋白(cold shock proteins,CSPs)基因,根据大肠杆菌(E.coli)CSPs蛋白保守氨基酸序列,借助生物信息学技术检索小麦EST序列,采用同源序列法拼接了小麦3个冷休克蛋白基因,并以小麦叶片总RNA为模板,采用RT-PCR对其进行扩增;同时,采用荧光定量PCR对3个基因的组织表达特性以及ABA、低温、高温、干旱和盐胁迫条件下的表达模式进行了研究。结果表明,小麦3个冷休克蛋白基因TaCSP1、TaCSP2和TaCSP3全长分别为290、374和377bp,各编码69、69、80个氨基酸残基。序列分析表明,TaCSP1、TaCSP2和TaCSP3之间氨基酸相似性为72.9%~84.3%,与大肠杆菌来源CSPs的相似性为40.0%~79.7%。荧光定量PCR表达谱分析显示,TaCSPs在抽穗期小麦的根、茎、叶和幼穗中均能表达。胁迫分析表明,3个冷休克蛋白基因在小麦幼根中的表达,在低温和ABA处理下,表现为早期诱导、后期抑制,在高温、干旱和盐胁迫下,则表现为早期抑制、后期有所恢复。克隆获得的小麦3个冷休克蛋白基因在根组织中强烈表达,并受ABA和冷胁迫诱导,推测其在小麦抵御逆境胁迫过程中发挥重要作用。     

橡胶树HbJAZ3突变体蛋白的原核表达及纯化

植物JAZ蛋白是茉莉酸信号调控途径的关键环节之一。HbJAZ3基因是橡胶树乳管细胞中编码JAZ蛋白的基因家族成员之一。本文采用原核表达和定点突变技术,构建了pET28a（+）-JAZ3、pET28a（+）-JAZ3-ZIM-mut（缺失ZIM结构域中TIFY基序）和pET28a（+）-JAZ3-Jas-mut（缺失Jas结构域的保守氨基酸FLEKRK）的His标签融合蛋白表达载体,并成功转化大肠杆菌Rosetta（DE3）。在37 ℃条件下,用1 mmol/L IPTG诱导2 h能够诱导目的蛋白以包涵体的形式大量表达。通过镍柱纯化了目的蛋白,获得了HbJAZ3及其ZIM结构域和Jas结构域的突变体蛋白,为进步一鉴定乳管细胞茉莉酸信号途径的关键环节打下了良好基础。     

小麦AQPs蛋白TaPIP1基因cDNA克隆及其表达分析

为探索小麦水通道蛋白在氮素处理过程中的生物学功能,以0.1%尿素处理6.0 h的周麦19幼根为材料,利用RTPCR方法克隆了小麦PIPs类型的水通道蛋白基因TaPIP1,并分析了该基因的组织表达特性及其在尿素处理过程中的表达特征。TaPIP1全长1 062 bp,包括61 bp的5′非翻译区,128 bp的 3′非翻译区和一个长度为873 bp、编码290个氨基酸的开放阅读框。序列分析表明,TaPIP1基因与已知小麦（GQ452384和AAM00368）、大麦（BAA23746,CAA54233和BAA23745）、水稻（BAA24016）和玉米（AAK26754 ,ACG39699,ACG37183,CAA57955和AAK26756）等单子叶植物来源的同类基因同源性较高,相似性为85.9%～99.3%。半定量RTPCR表达谱分析显示,TaPIP1在抽穗期小麦的根、茎和旗叶中均能表达。氮素处理下该基因表达分析结果显示,TaPIP1在萌发期小麦根系中的表达受尿素的诱导。     

小麦转录因子TaDREB6基因的克隆及鉴定

为了克隆小麦干旱应答基因,构建了干旱诱导的小麦cDNA文库,并从文库中分离了一个DREB类转录因子基因TaDREB6.序列分析表明,TaDREB6具有一个837bp的开放阅读框和242bp的3非编码区,推测的氨基酸序列中含有一个高度保守的AP2/EREBP结构域.采用该基因特异引物PCR技术对一套中国春缺体-四体材料进行扩增,将TaDREB6定位于3A染色体上.这是首次将一个小麦DREB基因定位在特定的染色体上.RT-PCR分析表明,TaDREB6基因受干旱胁迫诱导表达;亚细胞定位结果表明,TaDREB6-hGFP融合蛋白定位于细胞核中.上述结果说明,小麦TaDREB6基因编码的蛋白可能在细胞核内对干旱胁迫应答反应起调控作用.     

小麦Bowman-Birk型蛋白酶抑制因子的克隆表达及其抑制鱼糜凝胶软化的效果研究

为探究重组小麦(Triticum turgidum)Bowman-Birk蛋白酶抑制剂(BBI)抑制活性特征及其对鲢鱼肌原纤维蛋白自溶反应的抑制效果,采用TA克隆技术克隆小麦BBI成熟肽基因片段,并对原核表达的小麦BBI蛋白进行镍亲和层析纯化,利用SDS-PAGE、TSK-GEL G2000SWxl高效液相色谱检测诱导及纯化效果。在测定重组小麦BBI对胰蛋白酶的抑制活性的基础上,进一步通过SDS-PAGE分析了重组BBI对鲢鱼肌原纤维蛋白自溶反应的抑制作用。结果表明,经双酶切鉴定证实成功获得了BBI成熟肽基因片段,该基因与野生二粒小麦BBI相关基因(GenBank登录号EU346892.1)序列相似性为99.63%。目的蛋白在SDSPAGE及TSK-GEL G2000SWxl高效液相色谱分析中,分别表现为单一带和单一峰,产物得到有效纯化,分子量约10.5kD。该重组小麦BBI对胰蛋白酶有明显的抑制活性。添加量5μg·mg-1重组BBI可显著抑制鲢鱼肌原纤维蛋白(尤其是肌球蛋白重链)在pH 7.5,50℃下的自溶。     

小麦LEAsm基因及其启动子的克隆与功能研究

胚胎发育晚期丰富蛋白(LEA)是一个小的高亲水性的蛋白家族,该蛋白家族在逆境胁迫下大量积累,保护植物免受逆境胁迫。LEA蛋白可分为7组,其中重复的11-氨基酸基序是第3组LEA蛋白的特征。为深入分析第3组LEA蛋白在小麦响应逆境胁迫中的作用机制,利用芯片技术从小麦表达谱中筛选出一个渗透胁迫诱导表达的第3组LEA蛋白基因TaLEAsm,然后根据该基因序列设计引物筛选石麦15的BAC文库,获得1个含有该基因的BAC单克隆,以该BAC单克隆质粒为模板,通过BAC延伸测序克隆了TaLEAsm基因及其启动子序列,并对TaLEAsm序列特征、表达模式和启动子功能进行了初步分析。结果表明,TaLEAsm基因序列仅含有1个105bp的内含子,其开放读码框长675bp,编码224个氨基酸。TaLEAsm含有10个11-氨基酸重复序列,属于第3组LEA蛋白。低温、高盐和渗透胁迫均诱导TaLEAsm基因上调表达,但在根和叶中表达模式不同。在TaLEAsm基因起始密码子上游1 500bp序列中,预测含有14个逆境响应顺式元件。在拟南芥中,TaLEAsm基因启动子能够启动GUS基因表达,渗透胁迫诱导GUS基因明显上调表达。以上结果表明,TaLEAsm为小麦脱水响应基因,其启动子为渗透胁迫诱导启动子。     

茶树PPO融合蛋白真核表达载体的构建与表达

以迎霜品种茶树基因组为模板,利用高保真聚合酶pfu,通过PCR方法扩增得到茶树PPO基因的编码区序列。通过设计酶切引物将其成功融合至真核表达载体pPICZa中,构建出茶多酚氧化酶的融合蛋白真核表达载体pPICZa-PPO,并将其转化进入巴斯德毕赤酵母（Pichia pastoris）GS115中,成功筛选出多个阳性转化子。对阳性转化子进行甲醇诱导,采用Western-Blotting方法在培养液中成功检测到诱导表达的目的蛋白。酶活检测结果也显示诱导产物具有较高的相对酶活性,能够正确发挥酶蛋白的生物功能。     

簇毛麦新型低分子量谷蛋白亚基的分子克隆与序列分析

为发掘低分子量谷蛋白亚基（LMWGS）新的变异类型,通过设计引物和特异PCR扩增,从簇毛麦TA2127基因组DNA中得到14个LMWGS基因,GenBank登录号分别为HQ615147～HQ615160。序列分析表明,所有基因具有完整编码区,长度为831 bp和846 bp,可编码277和282个氨基酸残基,推导的氨基酸序列分析显示,14个基因编码的蛋白序列都缺少一段N端保守区,且N端序列是一种新的类型;在14个LMWGS基因中检测到19个SNPs和1个16 bp 的缺失。系统进化分析表明,簇毛麦LMWGS与ω醇溶蛋白和HMWGS亲缘关系相对较远,与普通小麦 Glu3的低分子谷蛋白基因进化关系相对较近。     

减少灌水次数对强筋小麦氮素积累转运和籽粒蛋白质含量的影响

为探究减少灌水次数对强筋小麦氮素积累、转运和籽粒蛋白质含量的影响,选用4个强筋小麦品种(津农7号、农优3号、师栾02-1和中麦998),在大田条件下设置3种灌水处理(CK:冬水+拔节水+开花水;W2:冬水+拔节水;W1:冬水),研究了减少灌水次数对强筋小麦植株各器官氮素的积累与转运、籽粒蛋白质及其组分的含量、蛋白质产量和籽粒产量的影响,并对协调强筋小麦籽粒蛋白质含量和产量的适宜灌水次数进行了探讨。结果表明,减少灌水次数后,强筋小麦营养器官氮素积累量明显降低,氮素转运量增加;籽粒蛋白质含量升高;蛋白质组分的变化表现为,清蛋白、球蛋白和谷蛋白含量升高,醇溶蛋白含量下降;籽粒产量显著下降,水分利用效率升高,但不同品种增幅不同。综上所述,减少灌水次数能促进强筋小麦营养器官的氮素向籽粒转运,使籽粒蛋白质含量升高,籽粒蛋白质品质得到改善。本试验条件下,W2处理(冬水+拔节水)可以有效协调强筋小麦产量和籽粒蛋白质含量的关系,同时提高水分利用效率。     

小麦叶片光呼吸与蛋白质积累关系的研究

以蛋白质含量较高的小麦品种ＰＨ８２－２－２、蛋白质含量较低的小麦品种泰山１号为试验材料,测定ＮａＨＳＯ３处理后小麦叶片的光合速率、光呼吸、小麦叶片硝酸还原酶活性、游离氨基酸含量以及籽粒蛋白质含量。结果表明,蛋白质含量较高的小麦叶片光呼吸高于蛋白质含量较低的小麦品种叶片光呼吸,而且不管光呼吸抑制与否均有此规律。小麦叶片光呼吸与蛋白质积累有密切的关系。     

Fractionation of cereal flour by sedimentation in non-aqueous systems. II. Rheological characterisation and baking performance of the protein fraction

A previously described method for the non-aqueous fractionation of cereal flours by sedimentation in non-aqueous solvents was carried out using flours of three wheat cultivars differing in baking performance, as well as one rye and one barley flour. The method was based on differences in the densities of starch (higher) and protein (lower). Thus, suspending finely milled flour in an inert solvent mixture with a density in between the densities of starch and protein yielded a sedimented starch fraction and a protein-rich fraction at the surface of the solvent. Further purification of this upper fraction provided a protein fraction, a middle fraction, and a lipid fraction. The protein fractions were examined by means of rheological methods such as micro-extension tests and creep-recovery tests. They also were reconstituted to standard flour with a protein content of 13.5%, which was used for micro-scale baking tests. Compared to aqueous isolated gluten, the hydrated protein fractions from wheat were much more extensible and had a lower resistance to extension. The baking performance of the wheat protein fractions was superior to gluten and comparable to the native wheat flours. The protein fraction from rye gave a wheat-like bread crumb, whereas the barley protein was not suited for bread making.     

Effects of tillage,crop rotation and nitrogen fertilization on wheat-grain quality grown under rainfed Mediterranean conditions

The grain quality of wheat is influenced by the protein content, which in turn depends on environmental conditions and cropping practices. We carried out a 3-year field study in a rainfed Mediterranean region on the effects of tillage, crop rotation and nitrogen fertilization on the grain quality of hard red spring wheat (Triticum aestivum) in terms of protein content, test weight and alveogram indices. Tillage treatments were no tillage (NT) and conventional tillage (CT). Crop rotations were wheat–sunflower (Helianthus annus L.) (WS), wheat–chickpea (Cicer arietinum L.) (WCP), wheat–fababean (Vicia faba L.) (WFB), wheat–fallow (WF) and continuous wheat (CW). Fertilizer nitrogen was used at three different rates: 50, 100 and 150 kg N ha⁻¹. A split–split plot design with four replicates was used. Grain protein content was found to be inversely proportional to rainfall during the growing season. The tillage method was also found to affect grain protein content, test weight and some grain quality indices. Through its effect on moisture and nitrate in the soil. The crop rotations that included a legume (WCP and WFB) had marked effects on wheat quality. The increased grain protein content and resulted in improved rheological properties of the dough (viz. a higher alveogram index and a more balanced tenacity/extensibility ratio). However, no differences due to N dilution in the plant were observed in the wettest year studied, which was also the highest yielding. Increasing the fertilizer N rate increased the grain protein content; this variable had the most marked influence on grain quality indices, though in the year that gave the highest yield the N dilution effect was observed. The many significant interactions among experimental variables reveal a close relationship among grain yield, protein content, grain quality and the wheat growth conditions. Specifically, the amount of rainfall and its distribution in the growing season strongly influenced N availability and uptake by the crop, as well as wheat-grain quality indices.     

Prediction of grain protein content in winter wheat (Triticum aestivum L.) using plant pigment ratio (PPR)

The applicability of the hyperspectral data from the canopy to the prediction of wheat grain quality was assessed for winter wheat. A training experiment and a validation experiment with contrasting nitrogen (N) levels and different cultivars were conducted, respectively, at different locations in Beijing, China. The wheat canopy spectral reflectance over 350–2500 nm, leaf N concentration and chlorophyll (Chl) concentration were measured at different growth stages, and the grain protein content was also determined after harvest. Eight vegetation indices (VIs) were compared relating to leaf N concentration, and the result indicated that the plant pigment ratio (PPR, (R550−R450)/(R550+R450)), a Chl-based index, was most applicable to predict wheat grain protein due to its significant correlation with leaf N concentration at the post-anthesis stage. Based on the relationships among PPR, leaf Chl concentration, leaf N concentration, and grain protein content, the statistical prediction models of grain protein content for Zhongyou9507 (a hard winter wheat) and Jingdong8 (a semi-hard winter wheat) were developed. The root mean square error (RMSE) of the 18 DAA (days after anthesis) model of Zhongyou9507 was 0.175; those of the anthesis model and the 11 DAA model of Jingdong8 were 0.238 and 0.982, respectively. Taking both the precision and accuracy into account, the 18 DAA model of Zhongyou9507 and the anthesis model of Jingdong8 were recommended to predict grain protein content for each cultivar. The result demonstrated that PPR could be used to assess grain quality of winter wheat.     

The influence of different nitrogen treatments on the size distribution of protein fractions in hard and soft wheat

The relative quantity of specific proteins, protein subunits, as well as amount and size-distribution of polymeric proteins in wheat kernels may vary due to environmental conditions. In this study, the effect of different nitrogen treatments on polymeric and monomeric proteins in wheat was determined. Two soft white biscuit wheat cultivars, a cracker wheat and a hard red bread wheat were planted under irrigation in a randomized complete block design with three replications in two successive years at six different nitrogen treatments. SE-HPLC was used to determine the amount of monomeric and polymeric proteins, and various quality characteristics were measured after harvesting. The large and small SDS-extractable polymeric proteins were not influenced by different N levels. There was a strong cultivar influence, where some cultivars had a larger reaction to N treatments than others. The later application (at flag leaf stage) of nitrogen did not increase the protein fractions. The total amount of N given seemed to have a greater influence on the protein fractions than the timing of the fertilizer application. The lowest N treatment consistently gave the lowest flour protein content value. There was a strong correlation between flour protein content and large monomeric proteins.     

小麦淀粉粒结合蛋白多态性及蛋白表达差异的研究

为了解小麦淀粉粒结合蛋白(Starch granule proteins,SGPs)的多样性及与淀粉品质性状的关系,对来自中国、墨西哥和埃及的149份春小麦材料淀粉粒结合蛋白进行SDS-PAGE电泳分析,探讨了淀粉粒结合蛋白多态性及表达差异。结果表明,供试材料的SGP组成存在多态性,149份材料中分离了10种主要的SGPs和56种组合带谱,其中SGP-g和SGP-e是小麦中出现频率最高的淀粉粒结合蛋白(出现频率均在90%以上),而绝大多数小麦材料(92.6%)中包含了7～9条SGPs条带。材料的地理来源与SGPs蛋白谱带聚类的结果并不完全一致。     

Quality of shear fractionated wheat gluten – Comparison to commercial vital wheat gluten

The functional properties of gluten obtained with a shear-induced separation process, recently proposed by Peighambardoust et al. (2008), are compared with a commercially available vital wheat gluten. Two tests were performed. First, a relatively strong wheat flour, Soissons, was enriched with gluten protein. The resulting dough was then evaluated on its kneading performance. Second, a weak flour, Kolibri, was enriched to evaluate the baking properties. The wheat flour enriched with gluten protein obtained via the shear-induced separation process (SCG) showed comparable to improved gluten functionality relative to commercial available vital wheat gluten protein (CVWG). The differences in functionality cannot be directly related to the composition as analyzed with SE-HPLC, because the composition of the gluten materials was rather comparable. The differences in functionality may therefore be related to the different drying techniques used or to the inherent mildness of the shear-induced separation technique.     

小麦籽粒发育时期Puroindolines蛋白与硬度的关系

为探讨Puroindolines蛋白的表达特点与籽粒硬度的关系,采用改进的SDS-PAGE凝胶分析了不同硬度小麦品种的籽粒在各个发育时期Puroindolines蛋白的表达.结果表明,不同硬度的小麦籽粒中总Puroindolines(PinA和PinB)蛋白的表达量差异不大,但与胚乳淀粉颗粒结合的Puroindolines蛋白量差异非常明显:在籽粒发育的不同时期,软质小麦籽粒淀粉粒表面结合的Puroindolines蛋白量显著高于硬质小麦;基因型同为野生型但硬度有差异的品种,籽粒较软的材料其淀粉粒表面结合的Puroindolines蛋白量也明显高于较硬的材料,说明该蛋白的结合特性是决定籽粒硬度的直接原因.结果还表明,胚乳中水溶性戊聚糖与籽粒硬度关系密切.     

休闲期耕作对旱地小麦籽粒蛋白质形成及其相关酶活性的影响

为给旱地小麦高产优质栽培提供理论依据,通过大田试验研究了旱地小麦休闲期不同时间深翻、深松对0～300 cm土壤蓄水量、小麦籽粒蛋白质形成及其与氮代谢相关酶活性关系的影响.结果表明,休闲期深翻或深松均可提高旱地小麦播前0～300 cm土壤蓄水量,且欠水年效果明显,以前茬小麦收获后45 d深翻效果较好.休闲期耕作均显著提高了小麦蛋白质产量.耕作时间对籽粒蛋白质含量的影响因降雨年型不同而异,欠水年的休闲期耕作均显著降低了籽粒蛋白质含量;丰水年在麦收后15d耕作显著降低了籽粒蛋白质含量,而麦收后45 d耕作则显著提高了籽粒蛋白质含量,尤其显著提高了醇溶蛋白和麦谷蛋白含量,从而改善了品质.此外,丰水年麦收后45 d耕作可提高花后旗叶和籽粒谷氨酰胺合成酶(GS)活性、旗叶和籽粒谷氨酸合成酶(GOGAT)活性,降低花后旗叶和籽粒谷氨酸脱氢酶(GDH)活性;籽粒蛋白质的累积在麦收后15d耕作条件下与籽粒GDH活性关系密切,而麦收后45 d耕作条件下与旗叶GS和GOGAT活性相关性较大.总之,旱地小麦休闲期耕作在不同降雨年型下均可起到良好的蓄水保墒作用,且欠水年效果较明显;耕作时间对土壤水分、小麦氮代谢酶活性、籽粒蛋白质及其组分含量具有较大的调控效应,休闲期雨后耕作有利于籽粒蛋白质形成,且深翻效果较好.