Isolation and expression analysis of genes encoding DNA methyltransferase in wheat (Triticum aestivum L.)

DNA methylation of cytosine residues, catalyzed by DNA methyltransferases, is suggested to play important roles in regulating gene expression and plant development. In this study, we isolated four wheat cDNA fragments and one cDNA with open reading frame encoding putative DNA methyltransferase and designated TaMET1, TaMET2a, TaMET2b, TaCMT, TaMET3, respectively. BLASTX searches and phylogenetic analysis suggested that five cDNAs belonged to four classes (Dnmt1, Dnmt2, CMT and Dnmt3) of DNA methyltransferase genes. TaMET2a encoded a protein of 376 aa and contained eight of ten conserved motifs characteristic of DNA methyltransferase. Genomic sequence of TaMET2a was obtained and found to contain ten introns and eleven exons. The expression analysis of the five genes revealed that they were expressed in developing seed, during germination and various vegetative tissues, but in quite different abundance. It was interesting to note that TaMET1 and TaMET3 mRNAs were clearly detected in dry seeds. Moreover, the differential expression patterns of five genes were observed between wheat hybrid and its parents in leaf, stem and root of jointing stage, some were up-regulated while some others were down-regulated in the hybrid. We concluded that multiple wheat DNA methyltransferase genes were present and might play important roles in wheat growth and development.     

Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.)

We have used a cDNA clone encoding a pathogen-induced putative wheat peroxidase to screen a genomic libary of wheat (Triticum aestivum L. cv. Cheyenne) and isolated one positive clone, lambda POX1. Sequence analysis revealed that this clone contains a gene encoding a putative peroxidase with a calculated pI of 8.1 which exhibits 58% and 83% sequence identity to the amino acid sequence of the turnip (Brassica rapa) peroxidase and a pathogen-induced putative wheat peroxidase, respectively. The two introns in the wheat gene are at the same positions as introns in the peroxidase genes of tomato and horseradish. Results of S1-mapping experiments suggest that this gene is neither pathogen-nor wound-induced in leaves but is constitutively expressed in roots.     

Silicon absorption by wheat (Triticum aestivum L.)

Although silicon (Si) is a quantitatively major inorganic constituent of higher plants the element is not considered generally essential for them. Therefore it is not included in the formulation of any of the solution cultures widely used in plant physiological research. One consequence of this state of affairs is that the absorption and transport of Si have not been investigated nearly as much as those of the elements accorded 'essential' status. In this paper we report experiments showing that Si is rapidly absorbed by wheat (Triticum aestivum L.) plants from solution cultures initially containing Si at 0.5 mM, a concentration realistic in terms of the concentrations of the element in soil solutions. Nearly mature plants (headed out) 'preloaded' with Si absorbed it at virtually the same rate as did plants grown previously in solutions to which Si had not been added. The rate of Si absorption increased by more than an order of magnitude between the 2-leaf and the 7-8 leaf stage, with little change thereafter. This revised version was published online in June 2006 with corrections to the Cover Date.     

The wheat (Triticum aestivum L.) leaf proteome

The wheat leaf proteome was mapped and partially characterized to function as a comparative template for future wheat research. In total, 404 proteins were visualized, and 277 of these were selected for analysis based on reproducibility and relative quantity. Using a combination of protein and expressed sequence tag database searching, 142 proteins were putatively identified with an identification success rate of 51%. The identified proteins were grouped according to their functional annotations with the majority (40%) being involved in energy production, primary, or secondary metabolism. Only 8% of the protein identifications lacked ascertainable functional annotation. The 51% ratio of successful identification and the 8% unclear functional annotation rate are major improvements over most previous plant proteomic studies. This clearly indicates the advancement of the plant protein and nucleic acid sequence and annotation data available in the databases, and shows the enhanced feasibility of future wheat leaf proteome research.     

Cloning and sequencing of cDNAs encoding a pathogen-induced putative peroxidase of wheat (Triticum aestivum L.)

We report here the complete amino acid sequence of a pathogen-induced putative peroxidase from wheat (Triticum aestivum L.) as deduced from cDNA clones representing mRNA from leaves infected with the powdery mildew fungus Erysiphe graminis. The protein consists of 312 amino acids, of which the first 22 form a putative signal sequence, and has a calculated pI of 5.7. Sequence comparison revealed that the putative wheat peroxidase is most similar to the turnip (Brassica rapa) peroxidase, with which it shares 57% identical and 13% conserved amino acids.     

The alpha-tubulin gene family in wheat (Triticum aestivum L.) and differential gene expression during cold acclimation.

The alpha-tubulins and beta-tubulins are the major constituents of microtubules, which have been recognized as important structural elements in cell growth and morphogenesis, and, recently, for their role in regulation and signal transduction. We have identified 15 full-length cDNAs for the members of the alpha-tubulin gene family in hexaploid bread wheat (Triticum aestivum L.). The genes were clustered into 5 homeologous groups of 3 genes. Representatives of the 5 homeologous groups were mapped to different chromosome arms, and the genome of origin was determined for each gene. Changes in mRNA levels were observed for the paralogous members of the gene family during cold acclimation. Three members of the family had initial decreases in mRNA levels in response to cold treatment, which were followed by increases, each with a different pattern of reinduction. One gene-family member showed increased mRNA for up to 14 d during cold acclimation and had decreased levels after 36 d of cold treatment; a fifth paralogous member of the gene family had slowly declining mRNA levels up to 36 d. Subtle differences in the level of gene expression among homeologs and large differences among paralogs were detected by comparing the relative abundance of wheat alpha-tubulin expressed sequence tags (ESTs) in public databases.     

Agrobacterium-mediated In-planta transformation of bread wheat (Triticum aestivum L.)

Journal of Plant Biochemistry and Biotechnology - Agrobacterium-mediated in-planta transformation method allows efficient plant transformation without tissue culture. In the present study, a tissue...     

Accelerated production of transgenic wheat (Triticum aestivum L.) plants

We have developed a method for the accelerated production of fertile transgenic wheat (Triticum aestivum L.) that yields rooted plants ready for transfer to soil in 8–9 weeks (56–66 days) after the initiation of cultures. This was made possible by improvements in the procedures used for culture, bombardment, and selection. Cultured immature embryos were given a 4–6 h pre-and 16 h post-bombardment osmotic treatment. The most consistent and satisfactory results were obtained with 30 g of gold particles/bombardment. No clear correlation was found between the frequencies of transient expression and stable transformation. The highest rates of regeneration and transformation were obtained when callus formation after bombardment was limited to two weeks in the dark, with or without selection, followed by selection during regeneration under light. Selection with bialaphos, and not phosphinothricin, yielded more vigorously growing transformed plantlets. The elongation of dark green plantlets in the presence of 4–5 mg/l bialaphos was found to be reliable for identifying transformed plants. Eighty independent transgenic wheat lines were produced in this study. Under optimum conditions, 32 transformed wheat plants were obtained from 2100 immature embryos in 56–66 days, making it possible to obtain R3 homozygous plants in less than a year.     

Suspension and protoplast culture of hexaploid wheat (Triticum aestivum L.)

Suspension cultures have been initiated from embryogenic callus of hexaploid wheat (Triticum aestivum L.). Most commonly, these suspensions are composed of callus-like clusters (up to 2 mm in diameter). Two rapidly-growing lines (MBE6 and C82d) have been obtained, which consist of smaller aggregates of cytoplasmic cells, and these have been maintained for more than 4 years. These lines show very limited morphogenetic capacity and only a single plantlet has been regenerated, from line MBE6, after 9 months in culture. Protoplasts isolated from line MBE6 are unable to divide, but protoplasts from line C82d consistently undergo sustained divisions to form callus or secondary cell suspensions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium     

Transformation of wheat (Triticum aestivum L.) through electroporation of protoplasts

Protoplasts isolated from embryogenic suspension cultures of wheat (Triticum aestivum cv. Hartog) were electroporated in the presence of plasmid pEmuGN and/or pEmuPAT, which contained the reporter gene gus and selectable marker gene bar, respectively. Under optimised electroporation conditions, up to 0.9% of viable protoplasts displayed gus activity two days after electroporation. To select for phosphinothricin (PPT) resistant colonies, electroporated protoplasts were incubated for six weeks in a medium containing 10 g/ml PPT. The cells surviving the selection were maintained as individual colonies on solid medium or as suspension cultures. More than 60% of these colonies exhibited tolerance to 40 g/ml PPT when tested 10 months after initial selection. To date, 57 green plants have been regenerated from these colonies and 24 have been transferred to soil. Southern blot analyses of colonies and plants, using the bar gene sequence as the probe, confirmed transformation of the cells. Positive PAT assays of both regenerated colonies and plants indicated the presence of the bar gene product. These results provide a basis for the establishment of routine procedures for transformation of wheat by direct gene transfer into protoplasts.Abbreviations gus -glucuronidase - PAT phosphinothricin N-acetyltransferase - PPT phosphinothricin - MS Murashige and Skoog medium     

Pollen-mediated gene flow in wheat (Triticum aestivum L.) in a semiarid field environment in Spain

Transgenic wheat (Triticum aestivum L.) varieties are being developed and field-tested in various countries. Concerns regarding gene flow from genetically modified (GM) crops to non-GM crops have stimulated research to estimate outcrossing in wheat prior to the release and commercialization of any transgenic cultivars. The aim is to ensure that coexistence of all types of wheat with GM wheat is feasible in accordance with current regulations. The present study describes the result of a field experiment under the semi-arid climate conditions of Madrid, Spain, at two locations (??La Canaleja?? and ??El Encin?? experimental stations) in Madrid over a 3-year period, from 2005 to 2007. The experimental design consisted of a 50?×?50?m wheat pollen source sown with wheat cultivars resistant to the herbicide chlortoluron (??Deganit?? and ??Castan?? respectively) and three susceptible receptor cultivars (??Abental??, ??Altria?? and ??Recital??) sown in replicated 1?×?1?m plots at different distances (0, 1, 3, 5, 10, 20, 40, 80 and 100?m) and four directions. Outcrossing rates were measured as a percentage of herbicide-resistant hybrids using an herbicide-screening assay. Outcrossing was greatest near the pollen source, averaging 0.029?% at 0?m distance at ??La Canaleja?? and 0.337?% at ??El Encin??, both below the 0.9?% European Union regulated threshold, although a maximum outcrossing rate of 3.5?% was detected in one recipient plot. These percentages declined rapidly as the distance increased, but hybrids were detected at different rates at distances of up to 100?m, the maximum distance of the experiment. Environmental conditions, as drought in 2004?C2005 and 2005?C2006, may have influenced the extent of outcrossing. These assays carried out in wheat under semi-arid conditions in Europe provide a more complete assessment of pollen-mediated gene flow in this crop.     

Plant regeneration from coleoptile tissue of wheat (Triticum aestivum L.)

Plant regeneration was achieved from coleoptile tissue of wheat (Triticum aestivum L. cv. Kharachia-65). Coleoptiles (1.0 - 3.5 cm long) were excised from 2- to 5-d-old seedlings and cultured on Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D - 0.5, 2.5, and 5.0 mg dm^-3). Cream, friable callus was obtained after 6 weeks of inoculation. This callus was sub-cultured on MS medium supplemented with 2,4-D (2.5 mg dm^-3) and 5 % coconut water. After 6 weeks of sub-culturing white, cream or pale, friable, nodular callus was obtained. Plant regeneration occurred when this callus was sub-cultured on MS medium supplemented with 0.2 mg dm^-3 1-naphthalene acetic acid + 1.0 mg dm^-3 6-benzylaminopurine. For rooting, regenerated shoots or plantlets were transferred on MS medium supplemented with 0.5 mg dm^-3 indole-3-acetic acid. Rooted plantlets were directly transferred into pots and grown under field conditions. Seed setting invariably occurred in all plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

水分胁迫对不同基因型小麦幼芽蛋白质表达和某些生理特性的影响

以抗旱小麦品种长武134和不抗旱小麦品种郑引1号为试材,采用-1.2 MPa PEG 6000处理种子,研究不同水分条件下小麦幼芽中蛋白质表达和部分生理特性的变化.聚丙烯酰胺凝胶电泳结果表明：水分胁迫可诱导抗旱品种幼芽产生分子量约39.5 kDa和23.0 kDa两种新蛋白亚基,不抗旱品种则无新亚基产生;在正常供水条件下,随着生育期延长,两品种中分子量为48.5 kDa的蛋白亚基表达量逐渐增强,因其对水分敏感且属于新发现蛋白,初步命名为水敏感蛋白.生理特性测定结果表明,水分胁迫条件下长武134的根芽比和相对含水量均高于郑引1号,而细胞膜相对透性和丙二醛含量则低于郑引1号.