Obtención de líneas de trigo con bajo contenido en epítopos responsables de la enfermedad celíaca mediante ARN de interferencia

En el presente trabajo se ha utilizado la transformación genética de trigo harinero para el silenciamiento de genes de gliadinas mediante ARN de interferencia (ARNi). En primer lugar se diseñaron vectores para el silenciamiento específico de las ¿-gliadinas. Las líneas transgénicas obtenidas mostraron silenciamientos de las ¿-gliadinas de entre el 77,4-85,5%, lo que produjo ligeros cambios en las propiedades del gluten y del almidón obtenidas por el Mixolab®. Sin embargo, para muchas de las líneas la supresión de las ¿-gliadinas no afectó a las propiedades de la masa, sugiriendo que las ¿-gliadinas no tienen un papel fundamental en la calidad harino-panadera. En segundo lugar se diseñaron nuevos vectores para el silenciamiento de todas las gliadinas, logrando reducciones de hasta un 95%. La calidad harino-panadera, medida mediante el test de sedimentación SDS, se mostró comparable con los controles no transformados para la mayoría de las líneas. A partir de la proteína del gluten se realizaron ensayos con clones de células T obtenidas de pacientes celíacos, obteniéndose una reducción muy significativa de la reactividad en las líneas transgénicas con silenciamiento de todas las gliadinas. La síntesis y deposición de las proteínas de almacenamiento en los cuerpos proteicos (CP) se estudió a lo largo del desarrollo del grano, observándose cambios en la forma y modo de agregación de los CP en las líneas con reducción de todas las gliadinas. También se observó un aumento en el número y tamaño de las inclusiones de los CP, en las que se ha descrito la presencia de triticinas. Este trabajo confirma que el silenciamiento basado en ARNi puede ser usado para la reducción específica de grupos de proteínas codificadas por familias multigénicas como son las ¿-gliadinas o los tres grupos de gliadinas (¿-, ¿-, y ¿-gliadinas). El silenciamiento de todas las gliadinas produjo una drástica reducción de los epítopos relacionados con la enfermedad celíaca, por lo que las harinas resultantes podrían emplearse para la elaboración de productos libres de gluten aptos para celíacos

Comparative genomic analysis and expression of the APETALA2-like genes from barley, wheat, and barley-wheat amphiploids

<p>Abstract</p> <p>Background</p> <p>The <it>APETALA2</it>-like genes form a large multi-gene family of transcription factors which play an important role during the plant life cycle, being key regulators of many developmental processes. Many studies in <it>Arabidopsis </it>have revealed that the <it>APETALA2 </it>(<it>AP2</it>) gene is implicated in the establishment of floral meristem and floral organ identity as well as temporal and spatial regulation of flower homeotic gene expression.</p> <p>Results</p> <p>In this work, we have cloned and characterised the <it>AP2</it>-like gene from accessions of <it>Hordeum chilense </it>and <it>Hordeum vulgare</it>, wild and domesticated barley, respectively, and compared with other <it>AP2 </it>homoeologous genes, including the Q gene in wheat. The <it>Hordeum AP2</it>-like genes contain two plant-specific DNA binding motifs called AP2 domains, as does the Q gene of wheat. We confirm that the <it>H. chilense AP2</it>-like gene is located on chromosome 5H^ch. Patterns of expression of the <it>AP2</it>-like genes were examined in floral organs and other tissues in barley, wheat and in tritordeum amphiploids (barley × wheat hybrids). In tritordeum amphiploids, the level of transcription of the barley <it>AP2</it>-like gene was lower than in its barley parental and the chromosome substitutions 1D/1H^chand 2D/2H^chwere seen to modify <it>AP2 </it>gene expression levels.</p> <p>Conclusion</p> <p>The results are of interest in order to understand the role of the <it>AP2</it>-like gene in the spike morphology of barley and wheat, and to understand the regulation of this gene in the amphiploids obtained from barley-wheat crossing. This information may have application in cereal breeding programs to up- or down-regulate the expression of <it>AP2</it>-like genes in order to modify spike characteristics and to obtain free-threshing plants.</p

Polinucleótido que comprende secuencias de gliadinas de trigo y su uso para silenciamiento mediante iARN

Peer reviewedConsejo Superior de Investigaciones CientíficasT3 Traducción de patente europe

Polinucleótido que comprende secuencias de gliadinas de trigo y su uso para silenciamiento mediante RNAi

Polinucleótido que comprende secuencias de gliadinas de trigo y su uso para silenciamiento mediante RNAi. La presente invención se refiere al silenciamiento especifico de las (alfa), (beta), (gamma) y ! (omega)- gliadinas de trigo duro y harinero mediante RNA de interferencia (ARNi) por medio del empleo de un polinucleótido que se transcribe a un hpRNA (hairpin RNA). Además, la presente invención también se refiere a un vector, célula, planta o semilla que comprenden el polinucleótido, cuya expresión se dirige de forma específica en tejidos concretos de las semillas de trigo mediante secuencias reguladoras de la expresión génica como por ejemplo, el promotor de un gen de -gliadinas o el promotor del gen que codifica para una D-hordeína.Peer reviewedConsejo Superior de Investigaciones Científicas (España)B1 Patente sin examen previ

Down-Regulating γ-gliadins in bread wheat leads to non-specific increases in other gluten proteins and has no major effect on dough gluten strength.

Background Gliadins are a major component of gluten proteins but their role in the mixing of dough is not well understood because their contribution to wheat flour functional properties are not as clear as for the glutenin fraction. Methodology/Principal Findings Transgenic lines of bread wheat with γ-gliadins suppressed by RNAi are reported. The effects on the gluten protein composition and on technological properties of flour were analyzed by RP-HPLC, by sodium dodecyl sulfate sedimentation (SDSS) test and by Mixograph analysis. The silencing of γ-gliadins by RNAi in wheat lines results in an increase in content of all other gluten proteins. Despite the gluten proteins compensation, in silico analysis of amino acid content showed no difference in the γ-gliadins silenced lines. The SDSS test and Mixograph parameters were slightly affected by the suppression of γ-gliadins. Conclusions/Significance Therefore, it is concluded that γ-gliadins do not have an essential functional contribution to the bread-making quality of wheat dough, and their role can be replaced by other gluten protein

Low-gluten, nontransgenic wheat engineered with CRISPR/Cas9

Coeliac disease is an autoimmune disorder triggered in genetically predisposed individuals by the ingestion of gluten proteins from wheat, barley and rye. The a-gliadin gene family of wheat contains four highly stimulatory peptides, of which the 33-mer is the main immunodominant peptide in patients with coeliac. We designed two sgRNAs to target a conserved region adjacent to the coding sequence for the 33-mer in the a-gliadin genes. Twenty-one mutant lines were generated, all showing strong reduction in a-gliadins. Up to 35 different genes were mutated in one of the lines of the 45 different genes identified in the wild type, while immunoreactivity was reduced by 85%. Transgene-free lines were identified, and no off-target mutations have been detected in any of the potential targets. The low-gluten, transgene-free wheat lines described here could be used to produce low-gluten foodstuff and serve as source material to introgress this trait into elite wheat varieties.Ministerio de Economía y Competitividad AGL2013-48946-C3-1-R ; AGL2016-80566-

Targeting of prolamins by RNAi in bread wheat: Effectiveness of seven silencing-fragment combinations for obtaining lines devoid of coeliac disease epitopes from highly immunogenic gliadins

Gluten proteins are responsible for the viscoelastic properties of wheat flour but also for triggering pathologies in susceptible individuals, of which coeliac disease (CD) and noncoeliac gluten sensitivity may affect up to 8% of the population. The only effective treatment for affected persons is a strict gluten-free diet. Here, we report the effectiveness of seven plasmid combinations, encompassing RNAi fragments from a-, c-, x-gliadins, and LMW glutenin subunits, for silencing the expression of different prolamin fractions. Silencing patterns of transgenic lines were analysed by gel electrophoresis, RP-HPLC and mass spectrometry (LC-MS/ MS), whereas gluten immunogenicity was assayed by an anti-gliadin 33-mer monoclonal antibody (moAb). Plasmid combinations 1 and 2 downregulated only c- and a-gliadins, respectively. Four plasmid combinations were highly effective in the silencing of x-gliadins and c-gliadins, and three of these also silenced a-gliadins. HMW glutenins were upregulated in all but one plasmid combination, while LMW glutenins were downregulated in three plasmid combinations. Total protein and starch contents were unaffected regardless of the plasmid combination used. Six plasmid combinations provided strong reduction in the gluten content as measured by moAb and for two combinations, this reduction was higher than 90% in comparison with the wild type. CD epitope analysis in peptides identified in LC-MS/MS showed that lines from three plasmid combinations were totally devoid of CD epitopes from the highly immunogenic a- and x-gliadins. Our findings raise the prospect of breeding wheat species with low levels of harmful gluten, and of achieving the important goal of developing nontoxic wheat cultivars

Molecular and Immunological Characterization of Gluten Proteins Isolated from Oat Cultivars That Differ in Toxicity for Celiac Disease

A strict gluten-free diet (GFD) is the only currently available therapeutic treatment for patients with celiac disease (CD). Traditionally, treatment with a GFD has excluded wheat, barley and rye, while the presence of oats is a subject of debate. The most-recent research indicates that some cultivars of oats can be a safe part of a GFD. In order to elucidate the toxicity of the prolamins from oat varieties with low, medium, and high CD toxicity, the avenin genes of these varieties were cloned and sequenced, and their expression quantified throughout the grain development. At the protein level, we have accomplished an exhaustive characterization and quantification of avenins by RP-HPLC and an analysis of immunogenicity of peptides present in prolamins of different oat cultivars. Avenin sequences were classified into three different groups, which have homology with S-rich prolamins of Triticeae. Avenin proteins presented a lower proline content than that of wheat gliadin; this may contribute to the low toxicity shown by oat avenins. The expression of avenin genes throughout the development stages has shown a pattern similar to that of prolamins of wheat and barley. RP-HPLC chromatograms showed protein peaks in the alcohol-soluble and reduced-soluble fractions. Therefore, oat grains had both monomeric and polymeric avenins, termed in this paper gliadin- and glutenin-like avenins. We found a direct correlation between the immunogenicity of the different oat varieties and the presence of the specific peptides with a higher/lower potential immunotoxicity. The specific peptides from the oat variety with the highest toxicity have shown a higher potential immunotoxicity. These results suggest that there is wide range of variation of potential immunotoxicity of oat cultivars that could be due to differences in the degree of immunogenicity in their sequences