Dataset Information

Artificial MicroRNA-Based Specific Gene Silencing of Grain Hardness Genes in Polyploid Cereals Appeared to Be Not Stable Over Transgenic Plant Generations.

ABSTRACT: Gene silencing by RNA interference is a particularly important tool in the study of gene function in polyploid cereal species for which the collections of natural or induced mutants are very limited. Previously we have been testing small interfering RNA-based approach of gene silencing in wheat and triticale. In this research, artificial microRNAs (amiRs) were studied in the same species and the same target genes to compare effectiveness of both gene silencing pathways. amiR cassettes were designed to silence Puroindoline a (Pina) and Puroindoline b (Pinb) hardness genes in wheat and their orthologues Secaloindoline a (Sina) and Secaloindoline b (Sinb) genes in triticale. Each of the two cassettes contained 21 nt microRNA (miR) precursor derived from conserved regions of Pina/Sina or Pinb/Sinb genes, respectively. Transgenic plants were obtained with high efficiency in two cultivars of wheat and one cultivar of triticale after using the Pinb-derived amiR vector for silencing of Pinb or Sinb, respectively. Lack of transgenic plants in wheat or very low transformation efficiency in triticale was observed using the Pina-derived amiR cassette, despite large numbers of embryos attempted. Silencing of Pinb in wheat and Sinb in triticale was highly efficient in the T1 generation. The transcript level of Pinb in wheat was reduced up to 92% and Sinb in triticale was reduced up to 98%. Moreover, intended silencing of Pinb/Sinb with Pinb-derived amiR cassette was highly correlated with simultaneous silencing of Pina/Sina in the same transgenic plants. High downregulation of Pinb/Pina genes in T1 plants of wheat and Sinb/Sina genes in T1 plants of triticale was associated with strong expression of Pinb-derived amiR. Silencing of the target genes correlated with increased grain hardness in both species. Total protein content in the grains of transgenic wheat was significantly lower. Although, the Pinb-derived amiR cassette was stably inherited in the T2 generation of wheat and triticale the silencing effect including strongly decreased expression of silenced genes as well as strong expression of Pinb-derived amiR was not transmitted. Advantages and disadvantages of posttranscriptional silencing of target genes by means of amiR and siRNA-based approaches in polyploid cereals are discussed.

SUBMITTER: Gasparis S

PROVIDER: S-EPMC5220083 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Artificial MicroRNA-Based Specific Gene Silencing of Grain Hardness Genes in Polyploid Cereals Appeared to Be Not Stable Over Transgenic Plant Generations.

Gasparis Sebastian S Kała Maciej M Przyborowski Mateusz M Orczyk Waclaw W Nadolska-Orczyk Anna A

Frontiers in plant science 20170109

Gene silencing by RNA interference is a particularly important tool in the study of gene function in polyploid cereal species for which the collections of natural or induced mutants are very limited. Previously we have been testing small interfering RNA-based approach of gene silencing in wheat and triticale. In this research, artificial microRNAs (amiRs) were studied in the same species and the same target genes to compare effectiveness of both gene silencing pathways. amiR cassettes were desig ...[more]

PMID: 28119710

Similar Datasets

Project description:A microarray analysis of wheat grain hardness For both the Heron and Falcon NIL, whole wheat heads were harvested at 6, 15 and 25 days post anthesis (DPA) from glass house grown material and stored at -80 OC. For the other wheat cultivars analysed, heads at approximately 9-10 DPA were used. Eight to ten developing caryopses at the same age and morphological stage of development were selected from a head to represent a sample. Seed collected from a different head represented a replicate sample. Total RNA was isolated from the whole seed following the method of Higgins et al. (1976) with the following modification to remove starch. After the lithium chloride precipitation, the RNA pellet was resuspended in 250 ?l water. Then 3.5 ?l of 3 M sodium acetate (pH 5.3) and 125 ?l ethanol were added and the sample was centrifuged for 10 minutes at 4 OC. The supernatant was transferred to a fresh tube and the RNA was ethanol-precipitated by the addition of 21.5 ?l of 3 M sodium acetate (pH 5.3) and 375 ?l ethanol. The recovery of RNA varied from 0.06-0.5 ?g RNA per mg of tissue and this was dependent on the developmental stage of the seeds used for extraction of the RNA. For the labelling procedure, 50 ?g of total RNA was used for both the Cy3 and Cy5 dyes (Amersham Pharmacia, UK), following the two-step labelling method of Schenk et al. (2000). The Cy3 control (hard wheat) and Cy5 sample (soft wheat) were swapped among the replicate experiments to minimise any bias in cDNA incorporation and photo-bleaching of the fluorescent dyes. The pre-hybridisation of the microarray slides, hybridisation of the target cDNA and subsequent washing of the slides to remove unbound target was performed as per the supplied protocol for the CMT-GAPSTM coated microscope slides (Corning USA). The slides were scanned with a GenePix 4000A microarray scanner (Axon Instruments, Union, CA, USA). The features were analysed using the GenePix Pro 4 software and unsatisfactorily segmented features were either manually adjusted or discarded to ensure the integrity of the data obtained.

Project description:The grain hardness index (HI) is one of the important reference bases for wheat quality and commodity properties; therefore, it is essential and useful to identify loci associated with the HI in wheat breeding. The grain hardness index of the natural population including 150 common wheat genotypes was measured in this study. The phenotypic data diversity of HI based on four environments and the best linear unbiased prediction (BLUP) was analyzed. The results showed that the grain HI of the natural population ranged from 15.00 to 83.00, the variation range was from 5.10% to 24.44%, and the correlation coefficient was 0.872-0.980. BLUP value was used to grade and assign the grain HI to hard wheat, mixed wheat, and soft wheat, and the assigned phenotypes were used for genome-wide association analysis. Two types of grain hardness index phenotypic values were used for genome-wide association analysis (GWAS) using a 55K SNP array. A total of five significant association loci (p < 0.001) were excavated, among which four loci could be detected in three or more environments. They were distributed on chromosomes 1A and 7D, and the phenotypic contribution rate was 7.52% to 10.66%. A total of 48 sites related to grain hardness were detected by the assignment method, among which five were stable genetic sites, distributed on chromosomes 1A(2), 3B(1), 4B(1), and 7D(1), with phenotypic contribution rates ranging from 7.63% to 11.12%. Of the five loci detected by the assignment method, two stable loci were co-located in the phenotypic mapping results of the hardness index. One of the loci was consistent with previous reports and located on chromosome 1A, and one locus was unreported on chromosome 7D. Therefore, it may be a feasible attempt to use the assignment method to conduct genome-wide association analysis of the grain hardness index. In this study, a total of five genetic loci for grain hardness stability were excavated, and two of the loci were located in the two phenotypic values, two of which were not reported.

Project description:Grain hardness is an important quality trait of cereal crops. In wheat, it is mainly determined by the Hardness locus that harbors genes encoding puroindoline A (PINA) and puroindoline B (PINB). Any deletion or mutation of these genes leading to the absence of PINA or to single amino acid changes in PINB leads to hard endosperms. Although it is generally acknowledged that hardness is controlled by adhesion strength between the protein matrix and starch granules, the physicochemical mechanisms connecting puroindolines and the starch-protein interactions are unknown as of this time. To explore these mechanisms, we focused on PINA. The overexpression in a hard wheat cultivar (cv. Courtot with the Pina-D1a and Pinb-D1d alleles) decreased grain hardness in a dose-related effect, suggesting an interactive process. When PINA was added to gliadins in solution, large aggregates of up to 13 μm in diameter were formed. Turbidimetry measurements showed that the PINA-gliadin interaction displayed a high cooperativity that increased with a decrease in pH from neutral to acid (pH 4) media, mimicking the pH change during endosperm development. No turbidity was observed in the presence of isolated α- and γ-gliadins, but non-cooperative interactions of PINA with these proteins could be confirmed by surface plasmon resonance. A significant higher interaction of PINA with γ-gliadins than with α-gliadins was observed. Similar binding behavior was observed with a recombinant repeated polypeptide that mimics the repeat domain of gliadins, i.e., (Pro-Gln-Gln-Pro-Tyr)8. Taken together, these results suggest that the interaction of PINA with a monomeric gliadin creates a nucleation point leading to the aggregation of other gliadins, a phenomenon that could prevent further interaction of the storage prolamins with starch granules. Consequently, the role of puroindoline-prolamin interactions on grain hardness should be addressed on the basis of previous observations that highlight the similar subcellular routing of storage prolamins and puroindolines.

Project description:The levels of 26 mycotoxins were determined in 147 samples of the grain of cereals cultivated in five regions of Poland during the 2014 growing season. The HPLC-HRMS (time-of-flight) analytical technique was used. An analytical procedure to simultaneously determine 26 mycotoxins in grain was developed, tested and verified. Samples from eastern and southern Poland were more contaminated with mycotoxins than the samples from northern and western Poland. Toxins produced by Fusarium fungi were the main contaminants found. Some deoxynivalenol (DON) was found in 100% of the tested samples of wheat (Osiny, Borusowa, Werbkowice), triticale, winter barley and oats, while the maximum permissible DON level (as defined in the EU Commission Regulation No. 1881/2006) was exceeded in 10 samples. Zearalenone (ZEN), DON metabolites and enniatins were also commonly found. The presence of mycotoxins in grain reflected the prevailing weather conditions during the plant flowering/earing stages, which were favorable for the development of blight. Among all investigated wheat genotypes, cv. Fidelius was the least contaminated, while Bamberka, Forkida and Kampana were the most contaminated. However, the single-factor ANOVA analysis of variance did not reveal (at a statistical significance level α = 0.05) any differences between levels of mycotoxins in individual genotypes. Triticale was the most contaminated grain among all of the tested varieties. ZEN, DON and the sum of 3-acetyldexynivalenol and 15-acetyldeoxynivalenol (3- and 15-ADON) were found in 100% of the tested triticale samples at concentrations within the 4-86, 196-1326 and 36-374 µg·kg(-1) range, respectively. Of particular concern was the fact that some "emerging mycotoxins" (enniatins) (in addition to commonly-known and legally-regulated mycotoxins) were also found in the tested triticale samples (enniatin B (Enn-B), enniatin B1 (Enn-B1), enniatin A-1 (Enn-A1), 100% of samples, and enniatin A (Enn-A), 70% of samples). Depending on the toxin, they were found at levels between 8 and 3328 µg·kg(-1).

Project description:Grain yield and protein content were determined for six wheat cultivars grown over three years at multiple sites and at multiple N-fertilizer inputs. Although grain protein was negatively correlated with yield, some grain samples had higher protein contents than expected based on their yields, a trait referred to as grain protein deviation (GPD). We used novel statistical approaches to calculate GPD across environment and to correlate gene expression in the developing caryopsis with this trait. The yield and protein content were initially adjusted for nitrogen fertilizer inputs, and then adjusted for yield (to remove the negative correlation) resulting in environmental corrected GPD. The transcriptome data for all samples were subjected to Principal Component Analysis (PCA) and ANOVA to identify individual Principal Components (PCs) correlating with GPD alone. Scores of the selected PCs significantly related to cultivar differences and GPD but not to the yield or protein content were identified as reflecting a multivariate pattern of gene expression related to genetic variation in GPD. Sets of genes significant for these PCs and hence GPD were identified as candidate genes determining cultivar differences in GPD. Microarray profiling has been used to identify the links between gene expression and grain protein content in 6 different varietes of wheat grown at 2 different sites, 3 N levels and during 3 growth seasons. Six UK cultivars (Istabraq , Hereward, Marksman, Cordiale, Malacca and Xi 19) were grown over three seasons (2008-9, 2009-10 and 2010-11) at Rothamsted . In the present publication, data from two sites are included: Rothamsted and RAGTResearch (Harpenden, UK) and at four other sites in the south-east of the UK (RAGT, Ickleton, Cambridge; Limagrain, Woolpit, Suffolk; Syngenta, Whittlesford, Cambridge; KWS-UK, Thriplow, Hertfordshire) in 2009-10 and 2010-11 only. Three replicate plots were grown at three N levels: 100kg/ha, (N100), 200kg/ha (N200) and 350 kg/ha (N350) (see Barraclough et al., 2010, Chope at al., 2014). Developing heads (10 per plot) were tagged and caryopses were harvested from the Rothamsted (2009, 2010 and 2011) and RAGT (2010 and 2011) sites at 21 days after anthesis (mid-grain filling) to measure gene expression using Affymetrix wheat microarrays giving a total of 161 samples. Six UK cultivars (Istabraq , Hereward, Marksman, Cordiale, Malacca and Xi 19) were grown over three seasons (2008-9, 2009-10 and 2010-11) at Rothamsted Research (Harpenden, UK) and at four other sites in the south-east of the UK (RAGT, Ickleton, Cambridge; Limagrain, Woolpit, Suffolk; Syngenta, Whittlesford, Cambridge; KWS-UK, Thriplow, Hertfordshire) in 2009-10 and 2010-11 only. Three replicate plots were grown at three N levels: 100kg/ha, (N100), 200kg/ha (N200) and 350 kg/ha (N350) (see Barraclough et al., 2010, Chope at al., 2014). Developing heads (10 per plot) were tagged and caryopses were harvested from the Rothamsted (2009, 2010 and 2011) and RAGT (2010 and 2011) sites at 21 days after anthesis (mid-grain filling) to measure gene expression using Affymetrix wheat microarrays giving a total of 161 samples.

Dataset Information

Artificial MicroRNA-Based Specific Gene Silencing of Grain Hardness Genes in Polyploid Cereals Appeared to Be Not Stable Over Transgenic Plant Generations.

Publications

Artificial MicroRNA-Based Specific Gene Silencing of Grain Hardness Genes in Polyploid Cereals Appeared to Be Not Stable Over Transgenic Plant Generations.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets