Molecular analysis of lipoxygenase (LOX) genes in common wheat and phylogenetic investigation of LOX proteins from model and crop plants

Lipoxygenase (LOX) proteins constitute an important class of lipid-hydrolyzing enzymes in cellular organisms. Here, we report a molecular analysis of LOX genes in common wheat (Triticum aestivum L.) and a more comprehensive phylogenetic investigation of the LOX proteins from higher plants. The full-length nucleotide sequences of two LOX genes (TaLOX1 and TaLOX2) from common wheat were isolated. TaLOX1 and TaLOX2 were assigned to the short arm of chromosome 4D and the long arm of chromosome 5D, respectively. TaLOX1 and TaLOX2 were both expressed in the developing grains of two common wheat varieties, indicating that they may contribute to the total LOX activity in common wheat seeds. A more extensive phylogenetic analysis conducted with 143 unique LOXs from model and crop plants revealed that plant LOXs could be classified into two subfamilies, which correlated with the absence (subfamily I) or presence (subfamily II) of a predicted chloroplast targeting peptide in the deduced LOX proteins. Our work provides new information on the LOX genes in common wheat and the phylogenetic relationships of higher plant LOXs, which may aid further functional and evolutionary studies of plant LOXs.     

干旱胁迫下小麦幼苗基因表达谱的cDNA AFLP分析

为了分离和识别小麦抗旱相关基因,给小麦抗旱节水育种提供依据,以既抗旱又具备高水分利用效率的新疆春小麦主栽品种新春6号为材料,采用cDNA-AFLP技术,分析了小麦在两叶一心期干旱胁迫条件下的诱导表达基因。通过253对引物组合的筛选,共得到748个干旱胁迫特异上调表达的转录衍生片段(TDF)。对其中22条片段进行克隆、测序、Blastx比对和功能分类分析的结果表明,有18个TDFs所推导的蛋白质序列与NCBI已有序列同源,功能涉及逆境胁迫反应、发育、信号转导、抗病蛋白、跨膜转运、能量代谢等方面;有4个TDFs在NCBI找到同源序列,但功能未知。     

小麦籽粒多酚氧化酶（PPO）检测方法的优化及其在育种中的应用

降低小麦中多酚氧化酶(PPO)活性,减缓面粉制品的褐化,是重要的育种目标之一。为了更好地服务于低PPO育种,本研究对检测PPO活性的原苯酚染色法进行了优化,更好地发挥了其鉴别力强、结果稳定、对种子活力伤害小等优点,便于育种者使用。苯酚染色和分子标记结果对比发现,染色结果可以很好地反映亲本(或高代)材料中PPO的基因型,特别在低PPO材料中吻合更好。对大量亲本和世代材料的籽粒染色发现,PPO不仅存在于种皮中,其活性还是由种皮基因型决定的,后代PPO性状表现出母性遗传和加性效应的特点,控制高PPO特性的两个主效基因之间具有明显的代偿作用。PPO性状遗传相对简单,纯合较快,F2以后籽粒的染色程度以单株为单位发生分离。尽管染色是针对种皮基因型的,但PPO基因的这些遗传特点和小麦的自交特性,使染色结果同样可以预测后代单株的分离前途。这一优化的籽粒染色法在低PPO育种中的有效性是可以肯定的。     

Characterization of Amaranthus cruentus L. seed proteins by 2-DE and LC/MS–MS: Identification and cloning of a novel late embryogenesis-abundant protein

Amaranth is taking great attention as an important cereal crop that could fulfill food requirements for the growing population, especially in developing countries. However, the protein composition of these seeds is not well known yet. We have used the proteomics tools to characterize amaranth seed proteome. About 400 proteins spots were resolved on two-dimensional gel electrophoresis (2-DE) and protein spots were analyzed by LC-MS/MS (liquid chromatography tandem mass spectrometry). Identified proteins were related to stress and defense responses, metabolic, respiratory, and oxide-reduction processes. One abundant spot was identified as a Late Embryogenesis Abundant (LEA) protein and the gene was cloned and characterized. The AcLEA cDNA contains a 418 bp open reading frame (ORF) encoding a polypeptide of 139 amino acids. Bioinformatics analysis showed that AcLEA belongs to LEAs Group 5. Proteomics is a powerful technique that could be used even in non-sequenced organisms such as amaranth. The obtained information reveals that amaranth seed, beyond the classical seed storage proteins, contains proteins related to protection against stress. The identification of these proteins opens the door to the application of new strategies to improve the quality of amaranth production.     

Definition of the low molecular weight glutenin subunit gene family members in a set of standard bread wheat (Triticum aestivum L.) varieties

Low-molecular-weight glutenin subunits (LMW-GS) are a class of seed storage proteins that play a major role in the determination of the viscoelastic properties of wheat dough. The LMW-GSs are encoded by multi-gene families at the Glu-A3, Glu-B3 and Glu-D3 loci, with more than 15 genes present in most bread wheat varieties. However, the genic profile associated with different alleles has not been clearly defined. Here, the LMW-GSs in a set of standard varieties were analyzed using molecular markers. In most cases, each Glu-3 allele was represented by a specific haplotype; however, some alleles were undistinguishable. The Glu-A3e and Glu-A3g alleles showed an identical marker haplotype, as did the alleles Glu-B3c and Glu-B3d, and Glu-B3f and Glu-B3ab. In contrast, two haplotypes among varieties designated Glu-D3c were differentiated. The marker profiles present at the Glu-D3 locus exhibited less variation compared to the genes at the Glu-A3 and Glu-B3 loci. Results show the potential of the LMW-GS gene marker system in the characterization of the LMW-GS alleles present in specific bread wheat varieties, and its reconciliation with protein-based nomenclature. This approach will advance the understanding of the contribution of each of the LMW-GS gene alleles in the control of the end-use quality.     

Variant high-molecular-weight glutenin subunits arising from biolistic transformation of wheat

Genetic transformation via the biolistic method has been used to introduce genes encoding natural and novel high-molecular-weight glutenin subunits (HMW-GS) into wheat. The appearance of new seed proteins of sizes not predicted by the transgene coding sequences was noted in some experiments. In this report, the identities of thirteen of these novel proteins were determined by tandem mass spectrometry (MS/MS). Seven different proteins larger than and two proteins smaller than the native protein were shown to contain peptides from 1Dx5. A novel protein found in some progeny of crosses between a transgenic plant and Great Plains winter wheats was larger than but contained several peptides from 1Dy10. In one line, a protein larger than and a protein smaller than HMW-GS each contained peptides from the N- and C-terminus of 1Dx5 and from the repeat region of 1Dy10. In a sixth transgenic line, the native Bx7 gene was apparently replaced by a gene that encodes a larger version of 1Bx7. The variant proteins accumulate in the polymeric protein fraction, indicating that they can form inter-molecular disulfide bonds. These results show that novel proteins found in some transformants are encoded by altered versions of either the transforming or endogenous HMW-GS genes.     

小麦PHO基因家族全基因组鉴定及表达分析

PHO基因家族主要参与磷酸盐的转运,在植物生长发育中起重要作用。为研究小麦PHO基因家族成员的潜在功能,本研究从小麦全基因组中鉴定PHO基因家族成员,并利用生物信息学手段对其基因结构特征、蛋白结构域、系统进化、顺式作用元件及表达特性进行分析。结果在小麦全基因组中共鉴定到12个PHO基因家族成员,所有成员均含有SPX和EXS结构域。系统进化、保守结构域和基因结构分析发现,小麦PHO蛋白与拟南芥PHO1-H1蛋白以及水稻PHO1蛋白亲缘关系较近;除TaPHO7、TaPHO10、TaPHO11和TaPHO12蛋白缺少部分基序外,其余小麦PHO蛋白均具有完整基序,且同一亚组内的成员具有相似的蛋白保守结构域和基因结构,说明PHO亚家族成员间高度保守。顺式作用元件分析发现,小麦PHO基因启动子区域含有与磷调控相关的激素诱导、光响应、低温响应等元件。基于RNA＿seq数据的组织特异性分析发现,大部分PHO基因在根中的表达量较高。qRT-PCR分析发现,低磷胁迫处理下磷高效小麦品种小偃54地下部分(根) TaPHO7和 TaPHO8基因的表达量显著高于对照。     

菲利普孢囊线虫侵染后小麦防卫相关UniGene表达特性

菲利普孢囊线虫(Heterodera filipjevi)是严重危害我国黄淮海地区小麦生产的重要病原物之一。为揭示该线虫侵染后合胞体建立早期病原与宿主细胞的互作机制,以H.filipjevi易感材料温麦19和抗性材料抗线2号为研究对象,应用real time PCR分析了线虫侵染后不同时段10个可能与防卫相关的UniGenes相对表达量的变化。结果表明,与细胞结构相关的基因UniGeneAK335102(GenBank ID,下同)在抗线2号中的表达量高于温麦19,而另一个与细胞结构相关的UniGeneDR737925在温麦19中的表达量高于抗线2号。与应激相关的一个UniGene JV948284在侵染后各时段,在温麦19中的表达量均远高于抗线2号。与抗性相关的UniGene JP233598在接种线虫3 d以后,在抗线2号中的表达量明显高于温麦19;UniGene JP852719(Ascorbate peroxidase)基因在抗、感材料中的表达量无明显差异;UniGene(CV767657)TIFY 3A-like protein在温麦19中的表达量随接种后时间延长而降低,而在抗线2号中的表达量仅在接种后3、9 d时的表达量相对较高。信号转导相关UniGeneCD87797与WRKY转录因子家族UniGeneEU665453,在温麦19中的表达量随接种后时间延长而逐渐增加,但在抗线2号中则逐渐降低。MYB转录因子UniGene JF951950的表达量在温麦19中接种早期较高,而在抗线2号中接种后期较高;另一MYB转录因子UniGeneAK330737(similarity to Os06g0258000)在抗线2号中的表达量明显高于温麦19。综上所述,在菲利普孢囊线虫侵染后的早期,10个可能与防卫相关的基因表达模式在抗性和易感材料中存在不同程度差异,此结果对揭示小麦对菲利普孢囊线虫抗、感的分子机制有一定启示。     

69份国外小麦品种(系)抗叶锈性鉴定及基因分析

为给抗叶锈病育种提供优异抗源,选用17个不同毒力的叶锈菌生理小种,对69份国外小麦品种（系）及36份已知抗病基因的近等基因系进行苗期抗叶锈病基因推导,并结合11个已知基因的分子标记进行标记检测;于2017-2018年度分别在河北保定试验田和河南周口黄泛区农场对69份国外小麦材料进行田间成株期抗叶锈性鉴定。结果发现,在69份国外小麦品种（系）中共检测到6个抗小麦叶锈病基因 （Lr1、Lr10、Lr19、Lr26、Lr34和Lr37）,携带苗期抗病基因Lr1、Lr10、Lr19和Lr26的分别有6、19、5和10份品种（系）,携带成株抗病基因Lr34和Lr37的分别有10和26份品种（系）,田间鉴定具有成株慢锈性的品种（系）共有46份。     

Molecular characterization of dimeric alpha-amylase inhibitor genes in wheat and development of genome allele-specific primers for the genes located on chromosome 3BS and 3DS

Alpha-amylase inhibitors are attractive candidates for the control of seed weevils as these insects are highly dependent on starch as an energy source. For weevil control, alpha-amylase inhibitors and their genes could be used to genetically engineer weevil resistant seeds. Thirty genes encoding dimeric alpha-amylase inhibitors were isolated from Triticum aestivum L. ‘Chinese Spring’ and characterized by nucleotide and amino acid sequence analysis. Eleven representative alpha-amylase inhibitor genes were identified, and the deduced amino acid sequences of these genes were of high coherence (95.1%). These inhibitors and others obtained from the wheat EST database were clustered into three groups, the genes from ‘Chinese Spring’ were present in each group. Specific primer sets were designed for each group, based on the SNPs of these genes, and the chromosome locations of each group of inhibitor genes investigated by amplification of the ‘Chinese Spring’ ditelosomic lines. There were two and one groups of inhibitor genes on chromosomes 3BS and 3DS, respectively, whereas no group of inhibitor genes was found on chromosome 3AS. Thus, the primer set for each group of inhibitor genes was genome allele-specific. The two known inhibitors, 0.53 and 0.19, were located on chromosomes 3BS and 3DS, respectively. The validity of the three genome allele-specific primer sets was confirmed by amplifications in 15 accessions of Triticum urartu, Triticum monococcum, Aegilops tauschii and Triticum dicoccoides. These results gave further support at the molecular level, that the 24 kDa dimeric alpha-amylase inhibitors in cultivated wheat are encoded by a multigene family.     

TaMBD2基因cDNA全长克隆及其在小麦叶片和种子中的表达

为探讨甲基结合蛋白(MBD)在小麦生长发育过程中的生物学功能,通过RACE技术克隆了小麦TaMBD2基因的cDNA全长,并分析了该基因在小麦叶片、种子发育及萌发过程中的表达特性。RACE克隆及序列分析表明,TaMBD2基因cDNA全长为1 511 bp,其中5 UTR 164 bp,3 UTR 405 bp,ORF 942 bp。对该基因编码氨基酸序列的分析发现,TaMBD2编码蛋白中包含有1个典型的甲基结合域和1个CW型锌指结构域;通过RT-PCR分析发现,TaMBD2基因在叶片中的表达随着小麦的生长发育而逐渐增强;在种子发育过程中,该基因在花后20和30 d时的表达量最高;在种子萌发过程的胚和胚乳中,该基因的表达水平变化不大。     

小麦DNA去甲基化酶基因全基因组鉴定及其在籽粒发育中的表达分析

DNA去甲基化酶（dMTase）是一种高度保守的表观遗传修饰因子,涉及许多生物学过程,包括生长发育、应激反应和次生代谢。本研究基于小麦基因组数据,对小麦 DNA去甲基化酶基因（TadMTase）进行了全面鉴定和生物信息学分析。结果表明,小麦基因组中包含18 个TadMTase基因,分布于小麦15条染色体上。系统进化分析将TadMTase分为 ROS、DML3、DML4 和 DML5等 4个亚家族,亚家族之间的TadMTase基因序列长度和内含子数量存在差异,但同一个系统进化树分支中的亚族成员具有高度相似的基因结构、保守 motifs 和结构域,为植物dMTase基因家族的直系同源基因,在进化方面具有保守性。亚细胞定位预测TadMTase均定位于细胞核中;通过与小麦祖先物种的进化及共线性分析,发现在小麦异源六倍体形成过程中存在部分TadMTase基因丢失;TadMTase基因家族启动子区域包含大量光信号、植物激素、胁迫响应和生长发育等相关顺式作用元件;转录组数据分析表明TadMTase基因在不同组织器官和籽粒发育不同时期表达模式不同,有一定的组织特异性。进一步RNA-Seq和荧光定量PCR分析表明TaROS1b-1A.1和TaROS1a-5A/D分别在籽粒的种皮和胚乳发育时期显著上调表达,且在强筋和弱筋小麦品种中表达存在差异。结果为TadMTase 基因在调控小麦籽粒生长发育及其品质形成中的调控机制提供参考。