四川小麦地方品种AS1643中α/β醇溶蛋白基因

用PCR方法从四川小麦地方品种AS1643中克隆到3个α/β-醇溶蛋白基因，即Gli-AS1643-1（GenBank No.DQ166376）、Gli-AS1643-2（GenBank No.DQ166377）和Gli-AS1643-3（GenBank No.DQ166378）。其中，Gli-AS1643-1和Gli-AS1643-2的编码区长度分别为873bp和852bp，可编码270和263个氨基酸残基的成熟蛋白。Gli-AS1643-3由于在编码区内有一个提前终止密码子，为不可编码成熟蛋白的假基因。序列比较显示Gli-AS1643-1、Gli-AS1643-2和 Gli-AS1643-3分别与GenBank中的α/β-醇溶蛋白基因具有较高的一致性，且序列结构非常相似。它们的N-端氨基酸序列与各种α-、β-、γ-和α/β-醇溶蛋白的基本一致，但与ω-醇溶蛋白和低分子量谷蛋白亚基的明显不同。N-端12肽串联重复紧密相关的5个脯氨酸框和类似于微卫星序列编码的2个多聚谷氨酰胺区域。在Gli-AS1643-2的N-端存在腹泻疾病活性序列，C-端含有12型腺病毒感染序列。Gli-AS1643-1、Gli-AS1643-2和Gli-AS1643-3各由6个保守的半胱氨酸残基形成3个分子内二硫键。     

尾状山羊草α-醇溶蛋白基因的克隆和序列分析

本研究利用一对α-醇溶蛋白基因的特异引物,从小麦近缘植物尾状山羊草Y46中克隆获得5个α-醇溶蛋白基因,与NCBI已提交的α-醇溶蛋白序列进行多重比对分析发现,本研究获得的α-醇溶蛋白基因与已知的小麦及其近缘植物中的α-醇溶蛋白基因序列具有较高的相似性,其编码的氨基酸序列存在一定的多态性;在潜在的致敏性上,在这5个α-醇溶蛋白序列中未发现任何已知的与乳糜泻病相关的抗原表位,这在小麦及其近缘植物中较为罕见;进化分析表明,来自C染色体组中的α-醇溶蛋白与来自M和U染色体组的α-醇溶蛋白具有较近的亲缘关系。     

Isolation and Analysis of α-Gliadin Gene Coding Sequences from Triticum durum

Three coding sequences of gliadins genes, designed as Gli2_Dul, Gli2_Du2 and Gli2_Du3, were isolated from the genomic DNA of Triticum durum accessions CItr5083. Gli2_Dul and Gli2_Du2 contain 945 and 864 bp, encoding the mature proteins with 314 and 287 amino acid residues, respectively. Gli2_Du3 is recognized as a pseudogene due to the stop codon occurring in the coding region. The pseudogenes, commonly occurring in gliadins family, are attributed to the single base change C→T. The amino acid sequences deduced from these gene sequences were characterized with the typical structure of α-gliadin proteins, including the toxic sequences （PSQQQP）. The peptide fraction PF（Y）PP（Q）is thought to be an extra unit of repetitive domain, slightly diverging from the previous report. Six cysteine residues were observed within two unique domains. Phylogenetic analysis showed Gli2_Du2 and Gli2_Du3 were closely related to the genes on chromosome 6A, whereas Gli2_Dul seems to be more homologous with the genes on chromosome 6B.     

野生二粒小麦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,用PCR方法从蛋白质含量低至13.17%的D81和高达27.20%的D42 2份野生二粒小麦(Triticumdicoccoides)中克隆得到2个LMW-GS基因序列LMW-D81和LMW-D42(GenBank上的序列号分别为FJ461691和FJ461690)。它们具有小麦低分子量谷蛋白基因的典型结构特征,其长度分别为1053bp和1011bp,并分别编码350和336个氨基酸残基的成熟蛋白。LMW-D42和LMW-D81的氨基酸序列估算分子量分别为38kDa和39kDa,说明二者均为C型亚基编码基因。LMW-D42和LMW-D81的N-末端序列都为METSHIP-,表明这2个C型亚基编码基因归属LMW-m型。同源性比对和聚类分析揭示,LMW-D81和LMW-D42均属于Glu-B3位点编码基因。LMW-D42和LMW-D81的核苷酸序列和推导的氨基酸序列一致性分别为93.94%和92.57%。与LMW-D81相比,LMW-D42除发生了22处间断性的碱基替换外,还存在一段42个碱基的缺失。对推导氨基酸序列进行的二级结构预测显示,LMW-D81和LMW-D42的蛋白质二级结构高度一致。其α-螺旋主要位于信号肽和C-末端,少量的α-螺旋和不规则卷曲构成了N-末端。大多数不规则卷曲位于重复区,仅有的一段β-折叠则出现在C-末端。同时,它们的编码区均具有分布一致的8个半胱氨酸残基,且第一和第七个半胱氨酸残基均位于无规则卷曲中。这些结构特点对小麦加工品质改良具有一定意义。     

华山新麦草α-醇溶蛋白基因的克隆及原核表达分析

 【研究目的】克隆华山新麦草（Psathyrostachys huashanica）的α-醇溶蛋白基因,并对其进行生物信息学分析,构建该基因的原核表达载体,在大肠杆菌中诱导表达融合蛋白。【方法】采用同源克隆法从华山新麦草基因组DNA中分离克隆出α-醇溶蛋白基因并进行序列分析,将克隆的华山新麦草α-醇溶蛋白基因Gli-Ns-5克隆到表达载体pET-28a (+)上,获得重组质粒pET28a-Gli-Ns转化大肠杆菌BL21 (DE3)并诱导表达。【结果】从华山新麦草基因组DNA中克隆了4个α-醇溶蛋白基因：Gli-Ns-2 (FJ713595)、Gli-Ns-3 (GQ139525)、Gli-Ns-4 (GQ139526)和Gli-Ns-5 (GQ139527)。序列分析表明,4条序列具有α-醇溶蛋白基因的典型结构特征,含有8个或9个半胱氨酸残基,序列FJ713595为假基因。利用所构建的大肠杆菌表达载体,经IPTG诱导,华山新麦草α-醇溶蛋白基因Gli-Ns-5(GQ139527)可在原核系统中特异性表达。Western-blot证实融合蛋白可成功表达。【结论】克隆了4个华山新麦草的α-醇溶蛋白基因序列,基因Gli-Ns-5(GQ139527)可在原核表达系统中成功表达,为小麦品质改良提供了新的候选基因。     

应用PCR技术研究中国特有小麦种子贮藏蛋白基因的遗传变异

利用Glu A1x、Glu B1x、Glu A3、Glu B3和Glu 1Dx5的特异性PCR引物 ,和位于 1BS染色体上的γ -醇溶蛋白和低分子谷蛋白 2对SSR标记 ,通过PCR的方法研究了 8份四川白麦子、1 4份云南铁壳麦、9份西藏半野生小麦和 9份新疆稻麦贮藏蛋白基因的遗传多样性。结果表明 ,γ -醇溶蛋白和低分子谷蛋白 2个SSR位点的遗传多样性较高 ,其次为Glu A3和Glu B3 ,而Glu Ax和Glu Bx的遗传多样性最低。在所有 4 0份供试材料均未扩增出Glu 1Dx5基因的特异DNA片段 ,说明这些小麦地方品种不含优质亚基 5的编码基因     

Isolation and Analysis of α-Gliadin Gene Coding Sequences from Triticum durum

Three coding sequences of gliadins genes, designed as Gli2_Du1, Gli2_Du2 and Gli2_Du3, were isolated from the genomic DNA of Triticum durum accessions CItr5083. Gli2_Du1 and Gli2_Du2 contain 945 and 864 bp, encoding the mature proteins with 314 and 287 amino acid residues, respectively. Gli2_Du3 is recognized as a pseudogene due to the stop codon occurring in the coding region. The pseudogenes, commonly occurring in gliadins family, are attributed to the single base change C → T. The amino acid sequences deduced from these gene sequences were characterized with the typical structure of α-gliadin proteins, including the toxic sequences (PSQQQP). The peptide fraction PF(Y)PP(Q)is thought to be an extra unit of repetitive domain, slightly diverging from the previous report. Six cysteine residues were observed within two unique domains. Phylogenetic analysis showed Gli2_Du2 and Gli2_Du3 were closely related to the genes on chromosome 6A, whereas Gli2_Du1 seems to be more homologous with the genes on chromosome 6B.     

Analysis of LMW-GS, α- and γ-Gliadin Gene Coding Sequences from Triticum macha

Novel LMW-GS （low molecular weight glutenin subunit）,α- and γ-gliadin from Triticum macha accessions were characterized via genomic PCR, which can do favor to improve the wheat quality. The complete coding regions of two α-gliadin, two γ-gliadin and two LMW-GS gene sequences, which designed as Gli-Mal, Gli-Ma2, Gli-Mrl, Gli-Mr2, Glu-LM1 and Glu-LM2, encoded the mature proteins with 307, 241, 348, 302, 474 and 377 amino acid residues, respectively. Gli-Mal and Gli-Ma2 were recognized as pseudogenes due to the in-frame stop codons. The amino acid sequences deduced from these gene sequences were characterized with the typical structure of α- or γ-gliadin or LMW- m type proteins with the exception of Gli-Ma2. Phylogenetic analysis showed Gli-Mal was closely related to those from T. aestivum, whereas Gli-Ma2 seemed to be more homologous with the gene sequences from Dasypyrum breviaristatum. Gli-Mrl was closely related to those from T. turgidum ssp. dicoccoides, while Gli-Mr2 was the nearest to those from T. aestivum. Glu-LM1 was closely related to those from Aegilops tauschii, whereas GIu-LM2 seemed to be more homologous with those from T. durum.     

Isolation and Sequence Analysis of HMW Glutenin Subunit 1Dy10. 1 Ecoding Gene from Xinjiang Wheat （Triticum petropavlovskyi Udacz. et Migusch）

A novel HMW glutenin subunit gene 1Dy10. 1 was isolated and characterized from Xinjiang wheat （Triticum petropavlovskyi. Udacz. et Migusch） accession Daomai 2. The complete open reading frame （ORF） of 1Dy10. 1 was 1 965 bp, encoding 655 amino acids. The numbers and distribution of cysteines in 1Dy 10.1 were similar to those of 1Dy10 and other y-type subunits. In the N-terminal of 1Dy 10.1, an amino acid was changed from L （leucine） to P （proline） at position 55. The repetitive domain of 1Dy10.1 differed from those of known HMW subunits by substitutions, insertions or/and deletions involving single or more amino acid residues. In the repetitive domain of subunit 1Dyl 0.1, the deletion of tripeptide GQQ in the consensus unit PGQGQQ resulted in the appearance of the motif PGQ that have not been observed in other known y-type HMW subunits. In comparison with the subunit 1Dy 12, a deletion of dipeptide GQ, which occurred in subunit IDy10, was also observed in subunit 1Dy10.1. The cloned IDylO. 1 gene had been successfully expressed in Escherichia coli, and the expressed protein had the identical mobility with the endogenous subunit IDy10.1 from seed.     

小麦品种“川农16”α-醇溶蛋白基因序列分析

 【目的】克隆和分析“川农16”醇溶蛋白基因,为其进一步遗传改良提供更多依据。【方法】根据已报道的α-醇溶蛋白基因序列设计引物,对小麦品种“川农16”总DNA进行PCR扩增得到约900 bp的DNA片段,分离纯化后连接到pMD18-T载体上,转化后筛选阳性克隆进行测序。【结果】获得4个不同的基因序列：Gli2-CN16-9、Gli2-CN16-12、Gli2-CN16-14和Gli2-CN16-6,GenBank登录号分别为DQ246446、DQ246447、DQ246448和DQ246449。其中,Gli2-CN16-9、Gli2-CN16-12和Gli2-CN16-14分别为861、870和900 bp,可分别编码286、289和299个氨基酸残基的成熟蛋白;而Gli2-CN16-6编码区长度为852 bp,由于存在2个提前终止密码子,不能编码有功能的成熟蛋白,为假基因。【结论】序列比较显示它们与α-醇溶蛋白基因有很高的一致性;与γ-和ω-醇溶蛋白基因差异明显。     

一个普通小麦γ-醇溶蛋白基因的分子标记

运用生物信息学方法,根据已知的黑麦75Kγ-黑麦碱DNA序列设计引物,对8份不同类型的普通小麦材料进行PCR扩增,均获得一条约400 bp的特异扩增带。对新中长和99L2的扩增带分别进行克隆测序,序列登录号为:DQ432029和DQ432030。分析表明,DQ432029和DQ432030序列完全一致,由377个碱基组成。BLAST分析发现,该序列与普通小麦γ-醇溶蛋白基因的同源性最高,相似性达92%,表明它是一个小麦γ-醇溶蛋白基因。同时,此序列与所有已知γ-醇溶蛋白基因序列存在显著差异,因而可以认为它是普通小麦γ-醇溶蛋白基因家族的一个新序列。所有不同类型的小麦材料都扩出一条同样大小的清晰、明亮带,此扩增带可以作为普通小麦该γ-醇溶蛋白新基因的特异分子标记。     

一种节节麦高分子量麦谷蛋白与小麦同源蛋白氨基酸序列的比较

对节节麦的一种y类高分子量麦谷蛋白基因进行了序列测定和比较。结果显示,该亚基与小麦A、B和D和节节麦D染色体编码的y型亚基的全蛋白氨基酸残基数目均不一致。6个y型基因的信号肽、N-端和C-端氨基酸残基数目均一致,但有个别氨基酸的替换。而重复区氨基酸残基数目均不一致,主要由于重复单元六肽和九肽的数目不等。在目前已知的D基因组编码的y型亚基中,Dy13t的重复区是最短的,它比Dy10少4个九肽,比Dy12少4个九肽,2个六肽,比Dy12t少4个九肽。由N-Calign序列所作的聚类分析将这6个y型亚基分为3支,A、B和D基因组编码的亚基各占一支。来源于D基因组的4个基因的一支,又可将来源于节节麦和小麦D基因组的2个基因分别聚类在一起。     

好气和厌氧条件下小麦秸秆的腐解特征研究

采用网袋培养法,研究在好气和厌氧条件,小麦秸秆腐解过程中碳、氮释放及纤维素、半纤维素和木质素的变化规律。结果表明:小麦秸秆在好气和厌氧条件的残留质量、碳和氮残留量均随培养时间的延长而降低,且呈前期(0~3个月)降解较快,而后(3~12个月)逐渐减缓的趋势。用一级动力学方程y=y0+a·e-kt对小麦秸秆在好气和厌氧条件的残留质量随时间变化进行拟合(决定系数R2均大于0.957),质量腐解半衰期分别为72.8和121.9d,腐解速率常数(k)分别为0.022 0和0.014 0/d。在好气条件,小麦秸秆中碳和氮元素释放速率常数分别是其在厌氧条件的1.79和1.67倍,小麦秸秆中碳和氮元素的释放率分别是其在厌氧条件的4.39和1.40倍,且在培养至1个月时处理之间呈显著性差异(P0.05)。小麦秸秆中的纤维素、半纤维素和木质素残留量均随培养时间延长总体呈下降趋势。以上结果表明,好气条件更有利于小麦秸秆碳氮元素的释放及纤维素、半纤维素和木质素的降解,从而更有利于小麦秸秆的降解。     

Influence of different nitrogen application on flour properties,gluten properties by HPLC and end-use quality of Korean wheat

This study was performed to identify how the different levels of nitrogen application affected the variances of gluten properties and end-use qualities and the differences of variances among Korean wheat cultivars. Protein and dry gluten content, SDS sedimentation volume and water absorption of Mixolab increased as nitrogen application increased. This ratio of the increase was higher in Korean wheat cultivars for bread than in Korean wheat cultivars for noodles and cookies. The proportion of(α+β)-gliadin measured by reversed-phase high-performance liquid chromatography(RP-HPLC) increased, but the proportion of ω-and γ-gliadin decreased as the protein content increased. The Korean wheat cultivars for bread showed a high proportion of(α+β)-gliadin increase, the Korean cultivars for noodles had a high proportion of γ-gliadin decrease and the Korean wheat cultivars for cookies had a high proportion of ω-gliadin decrease. However, there was no variation of the component in the proportion of glutenin component measured by RP-HPLC, even though the protein content was increased, but all of the protein fractions measured by size exclusion(SE)-HPLC were increased. The soluble monomeric protein showed a high proportion of Korean wheat cultivars for bread by the increase of protein content. Bread loaf volume increased by the increase of protein content but there were no variances in the ratio of increase among Korean wheat cultivars. The cookie diameter decreased with the increase of protein content, and this ratio of decrease was the highest in Korean wheat cultivars for cookies. The hardness of cooked noodles also increased by the increase of protein content but there were no variations in springiness and cohesiveness. The decrease proportion of ω-gliadin affected the increase of bread loaf volume, the hardness of cooked noodles, and the decrease of cookie diameter.     

郑麦366α-醇溶蛋白基因的克隆及生物信息学分析

为研究优质强筋小麦品种郑麦366α-醇溶蛋白的组成,应用简并引物进行PCR扩增,从郑麦366中扩增得到13条核苷酸序列,其中9条序列推导的氨基酸序列具有完整的开放阅读框。进一步分析显示,克隆的9个基因(分别命名为ZM366-1—ZM366-9)编码的蛋白质均具有α-醇溶蛋白的典型结构特征,根据T-细胞毒性抗原表位数目和多聚谷氨酰胺区特征分别将ZM366-1、ZM366-2定位到6A染色体,ZM366-3、ZM366-4定位到6D染色体,ZM366-5—ZM366-9定位到6B染色体。蛋白质二级结构预测显示,克隆的9个α-醇溶蛋白都仅含有α-螺旋结构,其中B基因组α-醇溶蛋白的α-螺旋含量明显高于其他基因组。同源系统进化树分析表明,克隆的9个α-醇溶蛋白具有明显的基因组特异性。     

Characterization of wheat monogenic lines with known Sr genes and wheat cultivars for resistance to three new races of Puccinia graminis f. sp. tritici in China

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a potentially devastating fungal disease of wheat worldwide. The present study was to evaluate the resistance of 42 wheat monogenic lines with known stem rust resistance (Sr) genes and 69 wheat cultivars to three new Pgt races (34C0MRGQM, 34C3MKGQM, and 34C6MTGSM) identified from aeciospores at the seedling and adult-plant stages. The phenotyping results revealed that monogenic lines harboring resistance genes Sr9e, Sr17, Sr21, Sr22, Sr26, Sr30, Sr31, Sr33, Sr35, Sr36, Sr37, Sr38, Sr47, SrTmp, and SrTt3 were effectively resistant to all three Pgt races at the seedling and adult-plant stages. In contrast, monogenic lines containing Sr5, Sr6, Sr7b, Sr9a, Sr9d, Sr9f, Sr9g, Sr9b, Sr16, Sr24, Sr28, and Sr39 were highly susceptible to these races at both seedling and adult-plant stages. The other lines with Sr8a, Sr10, Sr11, Sr13, Sr14, Sr15, Sr18, Sr20, Sr19, Sr23, Sr25, Sr27, Sr29, Sr32, and Sr34, displayed variable levels of resistance to one or two of the tested races. Seedling infection types (ITs) and adult-plant infection responses (IRs) indicated that 41 (59.4%) of the wheat cultivars showed high resistance to all the three races. Molecular marker analysis showed that four wheat culitvars likely carried Sr2, 20 wheat culitvars likely carried Sr31, 9 wheat culitvars likely carried Sr38, and none of the cultivars carried Sr24, Sr25, and Sr26. Our results provide a scientific basis for rational utilization of the tested Sr genes and wheat cultivars against these novel Pgt races.     

小麦地方品种半截芒Glu-B1位点HMW-GS基因的克隆及序列分析

高分子量谷蛋白亚基(HMW-GS)与小麦的品质特性紧密相关,挖掘小麦中HMW-GS新基因,对小麦品质改良具有重要意义。以小麦地方品种半截芒为材料,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDSPAGE)分析其HMW-GS组成,并设计特异性引物,利用PCR技术克隆其Glu-B1位点x型和y型亚基基因。序列分析表明,2个基因具有完整编码框,长度分别为2 367bp和2 106bp(GenBank登录号分别为KJ579439和KJ579440),编码789个氨基酸和702个氨基酸的蛋白,被命名为1Bx14*和1By15*。同源性搜索结果显示2条氨基酸序列与典型的HMW-GS有较高的同源性,且与普通小麦亚基1Bx14和1By15的同源性均为95%。系统进化树分析表明,1Bx14*和1By15*分别与1Bx14和1By15的遗传距离较近,聚类到同一分支上。     

一个提莫菲维小麦醇溶蛋白基因的克隆与序列分析

以提莫菲维小麦基因组DNA为模板,采用设计的小麦种子醇溶蛋白的保守引物进行PCR扩增,扩增产物插入pMD-18T载体,并转化到大肠杆菌DH5α中,对阳性克隆进行测序。结果表明,扩增产物长度为1 002 bp,包含一个完整的284个氨基酸的编码区,基因库登录号为DQ861428。序列比对表明,该序列为-αgliadin基因家系成员。利用-αgliadin基因的编码氨基酸序列建立系统树,分析表明该序列与栽培小麦供体物种一粒小麦的-αgliadin基因聚在一大类中,因而被定位在提莫菲维小麦的A染色体组上。