高大山羊草Aegilops longissima puroindoline b基因的克隆与表达分析

籽粒硬度是小麦加工品质的重要影响因素。puroindoline a（Pina）和puroindoline b（Pinb）是控制小麦籽粒硬度的主效基因。根据已报导的小麦Pinb基因的保守序列,设计合成了一对特异性引物,对高大山羊草Aegilops longissima（SS）的基因组DNA和胚乳RNA进行Pinb基因扩增、克隆、序列测定和表达分析,发现了一个新型Pinb等位基因。基因长360bp,编码119个氨基酸残基,对应于麦类作物Puroindoline B（PinB）成熟蛋白的结构区域,具有麦类作物Pinb基因特有的WPTKWWK的色氨酸结构域基因序列和10个半胱氨酸所形成的5个二硫键结构。与软粒小麦cv．Capitole的Pinb—D1a相比较,其核苷酸和氨基酸同源性分别为93．3％和92．4％。RT—PCR证实了Pinb基因在籽粒胚乳中的表达。研究结果表明,高大山羊草中包含着与小麦差异较大的籽粒硬度控制基因,为栽培小麦品质改良提供了丰富的遗传资源。     

牡山羊草Aegilops juvenalis Puroindoline b基因的克隆

Puroindoline a(Pina)和puroindoline b(Pinb)是控制小麦籽粒硬度的主效基因。根据已报道的小麦Pinb基因的保守序列,设计合成了一对特异性引物,对六倍体牡山羊草Aegilops juvenalis(UUMMDD)的基因组DNA和胚乳cDNA进行Pinb基因扩增、克隆和序列测定,发现了两个新型Pinb等位基因Pinb-allele-1和Pinb-allele-2。该基因全长360 bp,编码119个氨基酸残基。它编码的蛋白和麦类作物Puroindoline B(PinB)的成熟蛋白有非常高的同源性,具有麦类作物PinB蛋白所特有的WPTKWWK的色氨酸结构域和10个半胱氨酸所形成的5个二硫键结构。与软粒小麦cv.Capitole的Pinb-D1a相比较,其核苷酸同源性为93.1%、93.3%,氨基酸同源性为90.8%、92.4%。Pinb-allele-1和Pinb-allele-2分别含有11和9个氨基酸变异位点。RT-PCR证实了Pinb-allele-2基因在籽粒胚乳中的表达。Southern Blot分析结果表明,牡山羊草中含有两个拷贝的Pinb基因,其中包含着与小麦差异较大的籽粒硬度控制基因。     

粗山羊草(Aegilops tauschii)中Pinb基因的克隆和表达分析

puroindoline a(Pina)和puroindoline b(Pinb)是控制小麦籽粒硬度的主效基因。根据已报道的小麦Pinb基因的保守序列,设计合成了一对特异性引物,对粗山羊草Aegilops tauschii(DD)的基因组DNA进行Pinb基因扩增、克隆和序列分析,发现了一个新型Pinb等位基因。该基因长447 bp,编码148个氨基酸残基,具有麦类作物PinB蛋白所特有的WPTKWWK色氨酸结构域和10个半胱氨酸所形成的5个二硫键结构。与软粒小麦cv.Capitole的Pinb-D1a相比较,该基因含有14个氨基酸变异位点,其中包括一个紧邻色氨酸结构域的变异位点(Val66Phe),其核苷酸和氨基酸同源性分别为93.3%和90.5%。RT-PCR和Western Blot证实了Pinb基因在籽粒胚乳中的表达。Southern Blot分析结果表明,粗山羊草中Pinb基因为单拷贝。研究结果表明,粗山羊草中包含着与小麦差异较大的籽粒硬度控制基因,对此基因的进一步研究将加深对小麦籽粒硬度形成分子机制的了解。     

二角山羊草Pinb基因的克隆与表达分析

根据已报道的小麦Pinb基因的保守序列,设计合成了1对特异性引物,对二角山羊草(Aegilops bicornis,SS)的基因组DNA进行Pinb基因扩增、克隆、序列分析,发现了1个新型Pinb等位基因,基因长360bp,编码119个氨基酸残基,对应于麦类作物PinB成熟蛋白结构区域,具有其特有的WPTKWWK色氨酸结构域和10个半胱氨酸所形成的5个二硫键结构。与软粒小麦cv.Capitole的Pinb-D1a相比较,其核苷酸和氨基酸同源性分别为93.1%和91.6%。RT-PCR证实了Pinb基因在籽粒胚乳中的表达。研究结果表明,二角山羊草中包含着与小麦差异较大的籽粒硬度控制基因,为栽培小麦品质改良提供了丰富的遗传资源。     

中国春硬度基因的克隆与序列分析

目的：克隆、分析puroindoline a（Pina）、puroindoline b（Pinb）和grain softness protein（gsp）这三种控制小麦籽粒硬度的主要基因。方法：根据已报导的小麦3种基因的保守序列,设计合成了3对特异性引物,对六倍体软质小麦中国春基因组DNA进行基因扩增、克隆和序列测定,得到了3个基因的全长。结果：其ORF分别是447bp、447bp和495bp,编码的蛋白质全长分别为148aa、148aa和164aa。3种蛋白质都含有谷类作物所特有的19aa的信号肽,10个半胱氨酸所形成的5个二硫键结构,PINA和PINB蛋白有麦类作物所特有的富含色氨酸的结构域。结论：与粗山羊草的pina、pinb和gsp相比较,其核苷酸同源性为99．8％、95．8％和99．4％,氨基酸的同源性高达99．3％、96．6％和98．8％。3种与硬度相关的基因的分离与克隆丰富了小麦种质遗传资源库,为弄清影响小麦籽粒硬度的机制及其抗菌活性奠定了基础。     

长柔毛野豌豆编码甘油-3-磷酸酰基转移酶基因的克隆及序列分析

甘油－3－磷酸酰基转移酶（GPAT）基因与植物抗冷性密切相关。克隆到的长柔毛野豌豆（Vicia villosa)GPAT基因的编码区完整的cDNA片段长1377bp,编码458个氨基酸残基,与蚕豆（Vicia faba)和豌豆（Pissum sativum)比较,其核苷酸序列的同源性分别为94．1％和93．3％,氨基酸序列的同源性分别为96．9％和98．0％。     

小麦品质性状的免疫化学测定及不同近缘种属贮藏蛋白的免疫同源性

利用普通小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）高分子量谷蛋白亚基（ＨＭＷＧＳ）多克隆抗体和单克隆抗体,对小麦品质性状及不同麦类作物近缘种属的籽粒贮藏蛋白进行了免疫化学测定,以建立小麦品种性状的快速测定方法并研究不同麦类作物胚乳贮藏蛋白的免疫同源性。结果表明：抗原抗体反应与品质性状的相关性因抗体种类及品质性状的不同而不同,多抗略优于单抗,不同单、多抗间相差较大;籽粒蛋白质含量及干、湿面筋含量与吸附值相关程度较高,与沉淀值则中等,而面包性状相关性较差。多克隆抗体吸附值与籽粒蛋白质含量、湿面筋含量、干面筋含量、面包体积及面包比容的最大相关系数分别为０．７６２０、０．８９４２、０．８８７３、０．６１０３、０．４５９８和０．４７４４,单克隆抗体吸附值与其最大相关系数分别为０．７８３７、０．７７４５、０．７８２２、０．６８４１、０．６８７３和０．５９８２。小麦近缘种属籽粒贮藏蛋白与普通小麦１Ｄｙ１０亚基具有一定程度的免疫同源性,其中以黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）、斯卑尔脱（Ｔ．ｓｐｅｌｔａＬ．）、节节麦（ＡｅｇｉｌｏｐｓｓｑｕａｒｒｏｓａＬ．）及圆锥小麦（Ｔ．ｔｕｒｇｉｄｕｍＬ．）与其同源程度较高。     

玉米21kD富硫种子贮存蛋白的cDNA克隆及其序列分析

利用逆转录－聚合酶链式反应（ＲＴ－ＰＣＲ）方法从玉米掖单－２０开花后１０ｄ的叶片中分离到２１ｋＤ玉米种子贮存蛋白ｃＤＮＡ（Ｎ２１ＫＺＹ）,并进行了序列分析．其编码蛋白包含２１１个氨基酸,极其富含甲硫氨酸,高达２７％;其Ｎ端有一个２１个氨基酸的信号肽．Ｎ２１ＫＺＹ及其编码蛋白和Ｃｈｕｉ等人分离的该基因的基因组克隆及其编码蛋白的同源性分别为９５．１％和９０．５％;两者的编码蛋白与玉米１０ｋＤ醇溶蛋白极其相似,其中间多出一个５４氨基酸的肽段和一个６氨基酸的肽段,这表明它们可能是来源于同一个祖先基因,后来通过基因重排、缺失或不均等交换等过程而形成的不同的蛋白质．     

小麦面粉Puroindoline蛋白的提取与纯化

Puroindoline蛋白是小麦面粉中一种非常重要的蛋白质,不仅影响和决定了籽粒的硬度,而且有抗G^＋、G^-菌以及抗真菌的作用。用含4％TritonX-114、100mmol／L pH7．8Tris-HCl缓冲液处理小麦面粉来分离Puroindoline蛋白。经处理后得到的蛋白质混合溶液首先用分子筛葡聚糖G-75纯化,每个收集管内的组分经SDS-PAGE分析,分子量小于31kD的蛋白质组分被回收和集中,回收的蛋白质组分经PEG20000浓缩后,再用离子交换柱羧甲基纤维素（CM-23）进行纯化。其洗脱液分别是双蒸水和NaCl,梯度为0．05～0．7mol／L、8mmol／L pH5．5的MES缓冲液,回收只含15kD的蛋白质的组分,接着用PEG20000浓缩。最后冷冻干燥得到Puroindoline蛋白。     

小麦醇溶蛋白的研究

根据胚乳蛋白在不同溶荆中的溶解能力,可将小 麦胚乳蛋白分为四个组份臼”,其中可溶于70%酒精 的贮藏蛋白,称为小麦醇溶蛋白（gliadin),醇溶蛋白只 存在于禾本科植物种子中,它们构成了大部分具有重 要经济价值的谷物籽粒的主要贮藏蛋白〔,．〕。在小麦 中,它的含量约占籽粒总蛋白的一半。醇溶蛋白位于 胚乳的蛋白体内,主要存在于亚糊粉层,其富含疏水性 氨基酸,如亮氨酸, 氨酸、脯氨酸和谷酞胺等,缺乏亲 水性氨基酸,如赖氨酸、色氨酸和蛋氨酸等。氨基酸谱 的不平衡是使醇溶蛋白含量较高的小麦在实际应用上 受限制的一个重要原因。     

Recurrent deletions of puroindoline genes at the grain hardness locus in four independent lineages of polyploid wheat

Polyploidy is known to induce numerous genetic and epigenetic changes but little is known about their physiological bases. In wheat, grain texture is mainly determined by the Hardness (Ha) locus consisting of genes Puroindoline a (Pina) and b (Pinb). These genes are conserved in diploid progenitors but were deleted from the A and B genomes of tetraploid Triticum turgidum (AB). We now report the recurrent deletions of Pina-Pinb in other lineages of polyploid wheat. We analyzed the Ha haplotype structure in 90 diploid and 300 polyploid accessions of Triticum and Aegilops spp. Pin genes were conserved in all diploid species and deletion haplotypes were detected in all polyploid Triticum and most of the polyploid Aegilops spp. Two Pina-Pinb deletion haplotypes were found in hexaploid wheat (Triticum aestivum; ABD). Pina and Pinb were eliminated from the G genome, but maintained in the A genome of tetraploid Triticum timopheevii (AG). Subsequently, Pina and Pinb were deleted from the A genome but retained in the A(m) genome of hexaploid Triticum zhukovskyi (A(m)AG). Comparison of deletion breakpoints demonstrated that the Pina-Pinb deletion occurred independently and recurrently in the four polyploid wheat species. The implications of Pina-Pinb deletions for polyploid-driven evolution of gene and genome and its possible physiological significance are discussed.     

A novel chimeric low-molecular-weight glutenin subunit gene from the wild relatives of wheat Aegilops kotschyi and Ae. juvenalis: evolution at the Glu-3 loci

Four LMW-m and one novel chimeric (between LMW-i and LMW-m types) low-molecular-weight glutenin subunit (LMW-GS) genes from Aegilops neglecta (UUMM), Ae. kotschyi (UUSS), and Ae. juvenalis (DDMMUU) were isolated and characterized. Sequence structures showed that the 4 LMW-m-type genes, assigned to the M genome of Ae. neglecta, displayed a high homology with those from hexaploid common wheat. The novel chimeric gene, designed as AjkLMW-i, was isolated from both Ae. kotschyi and Ae. juvenalis and shown to be located on the U genome. Phylogentic analysis demonstrated that it had higher identity to the LMW-m-type than the LMW-i-type genes. A total of 20 single nucleotide polymorphisms (SNPs) were detected among the 4 LMW-m genes, with 13 of these being nonsynonymous SNPs that resulted in amino acid substitutions in the deduced mature proteins. Phylogenetic analysis demonstrated that it had higher identity to the LMW-m-type than the LMW-i-type genes. The divergence time estimation showed that the M and D genomes were closely related and diverged at 5.42 million years ago (MYA) while the differentiation between the U and A genomes was 6.82 MYA. We propose that, in addition to homologous recombination, an illegitimate recombination event on the U genome may have occurred 6.38 MYA and resulted in the generation of the chimeric gene AjkLMW-i, which may be an important genetic mechanism for the origin and evolution of LMW-GS Glu-3 alleles as well as other prolamin genes.     

Molecular basis of evolutionary events that shaped the hardness locus in diploid and polyploid wheat species (Triticum and Aegilops)

The Hardness (Ha) locus controls grain hardness in hexaploid wheat (Triticum aestivum) and its relatives (Triticum and Aegilops species) and represents a classical example of a trait whose variation arose from gene loss after polyploidization. In this study, we investigated the molecular basis of the evolutionary events observed at this locus by comparing corresponding sequences of diploid, tertraploid, and hexaploid wheat species (Triticum and Aegilops). Genomic rearrangements, such as transposable element insertions, genomic deletions, duplications, and inversions, were shown to constitute the major differences when the same genomes (i.e., the A, B, or D genomes) were compared between species of different ploidy levels. The comparative analysis allowed us to determine the extent and sequences of the rearranged regions as well as rearrangement breakpoints and sequence motifs at their boundaries, which suggest rearrangement by illegitimate recombination. Among these genomic rearrangements, the previously reported Pina and Pinb genes loss from the Ha locus of polyploid wheat species was caused by a large genomic deletion that probably occurred independently in the A and B genomes. Moreover, the Ha locus in the D genome of hexaploid wheat (T. aestivum) is 29 kb smaller than in the D genome of its diploid progenitor Ae. tauschii, principally because of transposable element insertions and two large deletions caused by illegitimate recombination. Our data suggest that illegitimate DNA recombination, leading to various genomic rearrangements, constitutes one of the major evolutionary mechanisms in wheat species.     

Relationship Between the Sequence and Function of Rubisco rbcL in Nucleo-Cytoplasmic Hybrid Wheat

Nucleo-cytoplasmic hybrid wheat NC4 is resistent to a number of stresses and produces high yield. It was obtained by crossing Aegilops squarrosa (♀) with Triticum aestivum ( ♂ ), and several back crossings. The rbcLs (the gene of large subunit of Rubisco ( rihulose-1, 5-bisphosphate carboxylase/oxygenase )) cloned from NC4 and T. aestivum have been sequenced, and the result showed that the rbcL of NC4 was originated from Ae. squarrosa. The ratio of carhoxylase activity to oxygenase activity, Vco2/Vo2, of hybrid NC4 Was lower than that of Ae. squarrosa, but higher than that of T. aestivum. This difference may be accounted for the higher yield of NC4 than that of T. aestivum. Sequence analysis showed that three nucleotides in the rbcL of NC4 which were different from those of T. aestivum, corresponded to the No. 14, 86 and 95 amino acid residues of rbcL.     

Creation and functional analysis of new Puroindoline alleles in Triticum aestivum

The Hardness (Ha) locus controls grain texture and affects many end-use properties of wheat (Triticum aestivum L.). The Ha locus is functionally comprised of the Puroindoline a and b genes, Pina and Pinb, respectively. The lack of Pin allelic diversity is a major factor limiting Ha functional analyses and wheat quality improvement. In order to create new Ha alleles, a 630 member M(2) population was produced in the soft white spring cultivar Alpowa using ethylmethane sulfonate mutagenesis. The M(2) population was screened to identify new alleles of Pina and Pinb. Eighteen new Pin alleles, including eight missense alleles, were identified. F(2) populations for four of the new Pin alleles were developed after crossing each back to non-mutant Alpowa. Grain hardness was then measured on F(2:3) seeds and the impact of each allele on grain hardness was quantified. The tested mutations were responsible for between 28 and 94% of the grain hardness variation and seed weight and vigor of all mutation lines was restored among the F(2) populations. Selection of new Pin alleles following direct phenotyping or direct sequencing is a successful approach to identify new Ha alleles useful in improving wheat product quality and understanding Ha locus function.     

小麦抗虫α-淀粉酶抑制因子成熟蛋白编码基因序列分析

对17份小麦和山羊草材料的小麦抗虫24kD-α-淀粉酶抑制因子成熟蛋白编码基因进行了分离克隆和序列分析。结果发现,在二倍体材料中α-淀粉酶抑制因子由单个基因编码,而在普通小麦中是以多拷贝的形式存在。从中得到17个24kD-α-淀粉酶抑制因子基因,其中2个来自普通小麦与1个来自粗山羊草的基因编码的抑制因子与WDAI-0-19的氨基酸序列完全相同,为同一蛋白。在普通小麦中得到1个编码蛋白质与WDAI-0-53十分相似的基因。序列分析表明,24kD-α-淀粉酶抑制因子成熟蛋白编码基因在序列大小与核酸组成上都十分相似,一致性达到91.2%。这说明小麦和山羊草中24kD-α-淀粉酶抑制因子基因可能起源于相同原始基因。