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eDNA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360bp,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ev．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一2分别含有11和9个氨基酸变异位点。RT—PCR证实了Pinb—allele一2基因在籽粒胚乳中的表达。SouthernBlot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。     

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。     

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。     

拟斯卑尔脱山羊草基因的克隆与表达研究

Avenin—like基因是近年来发现的一类新基因。根据小麦avenin-like基因的保守序列,设计合成了一对特异性引物,对拟斯卑尔脱山羊草（Aegilops spehoides,ss）的基因组DNA进行avenin-like基因扩增、克隆、序列测定和表达分析,发现了一个新型avenin—like基因。基因长855bp,编码284个氨基酸残基,分子量约为33kD。Souchern blot结果表明其属于多基因家族。RT—PCR证实了avenin-like基因在籽粒胚乳中特异性表达。其对应的氨基酸序列含有18个半胱氨酸残基,可以形成7对分子内二硫键。研究表明Avenin-like蛋白是一类新型的储藏蛋白。这为小麦加工品质的改良提供了理论依据和遗传资源。     

中国春小麦puroindoline a基因的克隆及其真核表达载体的构建

目的:puroindoline(pin)基因在控制麦类作物的籽粒硬度中起着重要作用。构建真核表达载体pcDNA3.1( )-pina-gfp,为pina基因在哺乳细胞中的表达提供基础。方法:利用PCR方法从中国春小麦基因组中克隆到了pina基因,将其插入真核表达载体pcDNA3.1( )-gfp,用PCR和酶切鉴定重组子。结果:PCR和酶切鉴定表明,所构建的真核表达重组质粒为pcDNA3.1( )-pina-gfp;将该片段克隆到pCF-T载体中,经测序验证,表明其为目的基因。结论:构建的pina基因真核表达载体pcDNA3.1( )-pina-gfp为pina基因在哺乳动物细胞中的表达提供了基础。     

披碱草属及其近源属植物种子胚乳细胞多样性研究

披碱草属不仅是小麦、大麦等作物的重要基因库,也是优良牧草的重要组成部分。胚乳是麦类作物种子的重要组成部分,其重量占籽粒重量在90%以上。胚乳特性是一个相对稳定的遗传性状,可以将胚乳特性作为植物分类和系统关系的一个指标。胚乳细胞特征作为植物的一类微形态特征,在麦类植物系统分类与进化研究中具有一定的价值。该研究对小麦族的6个披碱草属(Elymus)物种、2个拟鹅观草属(Pseudoroegneria)物种、1个大麦属(Hordeum)物种和2个冰草属(Agropyron)物种,共4属11份材料的胚乳细胞特征进行解剖观察并测量相关指标。结果表明:(1)不同属、种的植物种子胚乳细胞之间存在丰富的多样性,属间差异大于属内种间差异;(2)同样具有P染色体组的Agropyron cristatum和A.mongolicum与同样具有St染色体组的Pseudoroegneria libanotica和P.spicata各自在细胞形状和大小上的差异很小;(3)不同物种的胚乳细胞在大小、形状和数量上均表现出差异,但不能很好地反映属以及基因组间的差异,研究结果为揭示披碱草属植物的系统关系提供了胚乳细胞方面的证据,同时也为利用该属植物改良麦类作物品质积累资料。     

小麦胚乳细胞的分离及其淀粉体的计数

IsolationofEndosPermCellsandCalculationofTheirStarchGrainsinWheatPlants小麦胚乳重量占籽粒重的90／以上．胚乳细胞的分裂数目及其淀粉体的发育状况决定着籽粒的重量和品质卜‘。在胚乳细胞增殖期,如能增加胚乳细胞的分裂速度．就能增加产量k‘．因而分离胚乳细胞,观察胚乳细胞的发育技术与方法受到人们的重视）－’‘’。前人对胚乳细胞的计数大致有以下几种方法：（）根据胚乳体积与胚乳细胞体积之比推算”‘;（2）用纤维素酶分解胚乳．计数单位酶液中的细胞数）‘’;（3）先用纤维素分解胚乳后再用淀粉酶解离细胞中的淀粉．…     

Transferability of wheat microsatellites to diploid Aegilops species and determination of chromosomal localizations of microsatellites in the S genome.

Overall, 253 genomic wheat (Triticum aestivum) microsatellite markers were studied for their transferability to the diploid species Aegilops speltoides, Aegilops longissima, and Aegilops searsii, representing the S genome. In total, 88% of all the analyzed primer pairs of markers derived from the B genome of hexaploid wheat amplified DNA fragments in the genomes of the studied species. The transferability of simple sequence repeat (SSR) markers of the T. aestivum A and D genomes totaled 74%. Triticum aestivum-Ae. speltoides, T. aestivum-Ae. longissima, and T. aestivum-Ae. searsii chromosome addition lines allowed us to determine the chromosomal localizations of 103 microsatellite markers in the Aegilops genomes. The majority of them were localized to homoeologous chromosomes in the genome of Aegilops. Several instances of nonhomoeologous localization of T. aestivum SSR markers in the Aegilops genome were considered to be either amplification of other loci or putative translocations. The results of microsatellite analysis were used to study phylogenetic relationships among the 3 species of the Sitopsis section (Ae. speltoides, Ae. longissima, and Ae. searsii) and T. aestivum. The dendrogram obtained generally reflects the current views on phylogenetic relationships among these species.     

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.     

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.     

N-terminal sequence of omega-gliadins from Aegilops longissima. The origin of the genome of polyploid wheat

Using high-performance reversed phase liquid chromatography, the major components of omega-gliadins were isolated from four samples of Aegilops longissima. A high interspecific variability of Ae. longissima with regard to gliadin composition was demonstrated. The N-terminal sequences of omega-gliadins were determined. It was shown that omega-gliadins under study belong to the SRQ type earlier discovered in hexaploid wheat species and in Ae. squarrosa. It is supposed that this type of sequence is specific to the whole Aegilops genus. The N-terminal sequence of omega-gliadin of Ae. longissima was identified and its similarity to the alpha/beta-type sequence found in hexaploid wheat species was revealed. The data obtained are discussed in terms of the origin of polyploid wheat genomes.     

Seed-specific expression of the wheat puroindoline genes improves maize wet milling yields

The texture of maize ( Zea mays L.) seeds is important to seed processing properties, and soft dent maize is preferred for both wet-milling and livestock feed applications. The puroindoline genes ( Pina and Pinb ) are the functional components of the wheat ( Triticum aestivum L.) Hardness locus and together function to create soft grain texture in wheat. The PINs (PINA and PINB) are believed to act by binding to lipids on the surface of starch granules, preventing tight adhesion between starch granules and the surrounding protein matrix during seed maturation. Here, maize kernel structure and wet milling properties were successfully modified by the endosperm-specific expression of wheat Pins ( Pina and Pinb ). Pins were introduced into maize under the control of a maize γ- Zein promoter. Three Pina/Pinb expression positive transgenic lines were evaluated over two growing seasons. Textural analysis of the maize seeds indicated that the expression of PINs decreased adhesion between starch and protein matrix and reduced maize grain hardness significantly. Reduction in pressure required to fracture kernels ranged from 15.65% to 36.86% compared with control seeds. Further, the PINs transgenic maize seeds had increased levels of extractable starch as characterized by a small scale wet milling method. Starch yield was increased by 4.86% on average without negatively impacting starch purity. The development of softer maize hybrids with higher starch extractability would be of value to maize processors.