Yingyin Yao

Cloning and characterization of microRNAs from wheat (Triticum aestivum L.)

BackgroundMicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition. So far, identification of miRNAs has been limited to a few model plant species, such as Arabidopsis, rice and Populus, whose genomes have been sequenced. Wheat is one of the most important cereal crops worldwide. To date, only a few conserved miRNAs have been predicted in wheat and the computational identification of wheat miRNAs requires the genome sequence, which is unknown.ResultsTo identify novel as well as conserved miRNAs in wheat (Triticum aestivum L.), we constructed a small RNA library. High throughput sequencing of the library and subsequent analysis revealed the identification of 58 miRNAs, comprising 43 miRNA families. Of these, 35 miRNAs belong to 20 conserved miRNA families. The remaining 23 miRNAs are novel and form 23 miRNA families in wheat; more importantly, 4 of these new miRNAs (miR506, miR510, miR514 and miR516) appear to be monocot-specific. Northern blot analysis indicated that some of the new miRNAs are preferentially expressed in certain tissues. Based on sequence homology, we predicted 46 potential targets. Thus, we have identified a large number of monocot-specific and wheat-specific miRNAs. These results indicate that both conserved and wheat-specific miRNAs play important roles in wheat growth and development, stress responses and other physiological processes.ConclusionThis study led to the discovery of 58 wheat miRNAs comprising 43 miRNA families; 20 of these families are conserved and 23 are novel in wheat. It provides a first large scale cloning and characterization of wheat miRNAs and their predicted targets.

Diverse set of microRNAs are responsive to powdery mildew infection and heat stress in wheat (Triticum aestivum L.)

BackgroundMicroRNAs (miRNAs) are a class of small non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition. MiRNAs can have large-scale regulatory effects on development and stress response in plants.ResultsTo test whether miRNAs play roles in regulating response to powdery mildew infection and heat stress in wheat, by using Solexa high-throughput sequencing we cloned the small RNA from wheat leaves infected by preponderant physiological strain Erysiphe graminis f. sp. tritici (Egt) or by heat stress treatment. A total of 153 miRNAs were identified, which belong to 51 known and 81 novel miRNA families. We found that 24 and 12 miRNAs were responsive to powdery mildew infection and heat stress, respectively. We further predicted that 149 target genes were potentially regulated by the novel wheat miRNA.ConclusionsOur results indicated that diverse set of wheat miRNAs were responsive to powdery mildew infection and heat stress and could function in wheat responses to both biotic and abiotic stresses.

Deep sequencing identifies novel and conserved microRNAs in peanuts (Arachis hypogaea L.)

BackgroundMicroRNAs (miRNAs) are a new class of small, endogenous RNAs that play a regulatory role in the cell by negatively affecting gene expression at the post-transcriptional level. miRNAs have been shown to control numerous genes involved in various biological and metabolic processes. There have been extensive studies on discovering miRNAs and analyzing their functions in model species, such as Arabidopsis and rice. Increasing investigations have been performed on important agricultural crops including soybean, conifers, and Phaselous vulgaris but no studies have been reported on discovering peanut miRNAs using a cloning strategy.ResultsIn this study, we employed the next generation high through-put Solexa sequencing technology to clone and identify both conserved and species-specific miRNAs in peanuts. Next generation high through-put Solexa sequencing showed that peanuts have a complex small RNA population and the length of small RNAs varied, 24-nt being the predominant length for a majority of the small RNAs. Combining the deep sequencing and bioinformatics, we discovered 14 novel miRNA families as well as 75 conserved miRNAs in peanuts. All 14 novel peanut miRNAs are considered to be species-specific because no homologs have been found in other plant species except ahy-miRn1, which has a homolog in soybean. qRT-PCR analysis demonstrated that both conserved and peanut-specific miRNAs are expressed in peanuts.ConclusionsThis study led to the discovery of 14 novel and 22 conserved miRNA families from peanut. These results show that regulatory miRNAs exist in agronomically important peanuts and may play an important role in peanut growth, development, and response to environmental stress.

Identification and characterization of wheat long non-protein coding RNAs responsive to powdery mildew infection and heat stress by using microarray analysis and SBS sequencing

BackgroundBiotic and abiotic stresses, such as powdery mildew infection and high temperature, are important limiting factors for yield and grain quality in wheat production. Emerging evidences suggest that long non-protein coding RNAs (npcRNAs) are developmentally regulated and play roles in development and stress responses of plants. However, identification of long npcRNAs is limited to a few plant species, such as Arabidopsis, rice and maize, no systematic identification of long npcRNAs and their responses to abiotic and biotic stresses is reported in wheat.ResultsIn this study, by using computational analysis and experimental approach we identified 125 putative wheat stress responsive long npcRNAs, which are not conserved among plant species. Among them, some were precursors of small RNAs such as microRNAs and siRNAs, two long npcRNAs were identified as signal recognition particle (SRP) 7S RNA variants, and three were characterized as U3 snoRNAs. We found that wheat long npcRNAs showed tissue dependent expression patterns and were responsive to powdery mildew infection and heat stress.ConclusionOur results indicated that diverse sets of wheat long npcRNAs were responsive to powdery mildew infection and heat stress, and could function in wheat responses to both biotic and abiotic stresses, which provided a starting point to understand their functions and regulatory mechanisms in the future.

TamiR159 Directed Wheat TaGAMYB Cleavage and Its Involvement in Anther Development and Heat Response

In Arabidopsis and rice, miR159-regulated GAMYB-like family transcription factors function in flower development and gibberellin (GA) signaling in cereal aleurone cells. In this study, the involvement of miR159 in the regulation of its putative target TaGAMYB and its relationship to wheat development were investigated. First, we demonstrated that cleavage of TaGAMYB1 and TaGAMYB2 was directed by miR159 using 5′-RACE and a transient expression system. Second, we overexpressed TamiR159, TaGAMYB1 and mTaGAMYB1 (impaired in the miR159 binding site) in transgenic rice, revealing that the accumulation in rice of mature miR159 derived from the precursor of wheat resulted in delayed heading time and male sterility. In addition, the number of tillers and primary branches in rice overexpressing mTaGAMYB1 increased relative to the wild type. Our previous study reported that TamiR159 was downregulated after two hours of heat stress treatment in wheat (Triticum aestivum L.). Most notably, the TamiR159 overexpression rice lines were more sensitive to heat stress relative to the wild type, indicating that the downregulation of TamiR159 in wheat after heat stress might participate in a heat stress-related signaling pathway, in turn contributing to heat stress tolerance.

Wheat Dof transcription factor WPBF interacts with TaQM and activates transcription of an alpha-gliadin gene during wheat seed development

Wheat prolamin-box binding factor (WPBF), a DOF transcription factor previously was isolated from wheat endosperm and suggested to function as an activator of prolamin gene expression during seed development. In this study, we showed that WPBF is expressed in all wheat tissues analyzed, and a protein, TaQM, was identified from a wheat root cDNA library, to interact with the Dof domain of WPBF. The specific interaction between WPBF and TaQM was confirmed by pull-down assay and bimolecular fluorescence complementation (BiFC) experiment. The expression patterns of TaQM gene are similar with that of WPBF. The GST-WPBF expressed in bacteria binds the Prolamin box (PB) 5′-TGTAAAG-3′, derived from the promoter region of a native alpha-gliadin gene encoding a storage protein. Transient expression experiments in co-transfected BY-2 protoplast cells demonstrated that WPBF trans-activated transcription from native alpha-gliadin promoter through binding to the intact PB. When WPBF and TaQM are co-transfected together the transcription activity of alpha-gliadin gene was six-fold higher than when WPBF was transfected alone. Furthermore, the promoter activities of WPBF gene were observed in the seeds and the vascular system of transgenic Arabidopsis, which was identical to the expression profiles of WPBF in wheat. Hence, we proposed that WPBF functions not only during wheat seed development but also during other growth and development processes.

Whole-genome discovery of miRNAs and their targets in wheat ( Triticum aestivum L.)

BackgroundMicroRNAs (miRNAs) are small, non-coding RNAs playing essential roles in plant growth, development, and stress responses. Sequencing of small RNAs is a starting point for understanding their number, diversity, expression and possible roles in plants.ResultsIn this study, we conducted a genome-wide survey of wheat miRNAs from 11 tissues, characterizing a total of 323 novel miRNAs belonging to 276 families in wheat. A miRNA conservation analysis identified 191 wheat-specific miRNAs, 2 monocot-specific miRNAs, and 30 wheat-specific variants from 9 highly conserved miRNA families. To understand possible roles of wheat miRNAs, we determined 524 potential targets for 124 miRNA families through degradome sequencing, and cleavage of a subset of them was validated via 5′ RACE. Based on the genome-wide identification and characterization of miRNAs and their associated target genes, we further identified 64 miRNAs preferentially expressing in developing or germinating grains, which could play important roles in grain development.ConclusionWe discovered 323 wheat novel miRNAs and 524 target genes for 124 miRNA families in a genome-wide level, and our data will serve as a foundation for future research into the functional roles of miRNAs in wheat.

Dynamic Expression of Imprinted Genes Associates with Maternally Controlled Nutrient Allocation during Maize Endosperm Development

Genomic imprinting refers to the differential expression of parental alleles in a parent-of-origin manner. Through a genome-wide identification of the imprinted genes in hybrid maize endosperm, this work provides evidence that the allele-specific expression status of the most imprinted genes is subject to dynamic change associated with different developmental events in the maize endosperm. In angiosperms, the endosperm provides nutrients for embryogenesis and seed germination and is the primary tissue where gene imprinting occurs. To identify the imprintome of early developing maize (Zea mays) endosperm, we performed high-throughput transcriptome sequencing of whole kernels at 0, 3, and 5 d after pollination (DAP) and endosperms at 7, 10, and 15 DAP, using B73 by Mo17 reciprocal crosses. We observed gradually increased expression of paternal transcripts in 3- and 5-DAP kernels. In 7-DAP endosperm, the majority of the genes tested reached a 2:1 maternal versus paternal ratio, suggesting that paternal genes are nearly fully activated by 7 DAP. A total of 116, 234, and 63 genes exhibiting parent-specific expression were identified at 7, 10, and 15 DAP, respectively. The largest proportion of paternally expressed genes was at 7 DAP, mainly due to the significantly deviated parental allele expression ratio of these genes at this stage, while nearly 80% of the maternally expressed genes (MEGs) were specific to 10 DAP and were primarily attributed to sharply increased expression levels compared with the other stages. Gene ontology enrichment analysis of the imprinted genes suggested that 10-DAP endosperm-specific MEGs are involved in nutrient uptake and allocation and the auxin signaling pathway, coincident with the onset of starch and storage protein accumulation.

Characterization and expression of 42 MADS-box genes in wheat (Triticum aestivum L.)

MADS-box genes form a large family of transcription factors and play important roles in flower development and organ differentiation in plants. In this study, 42 wheat cDNAs encoding putative MADS-box genes were isolated. BLASTX searches and phylogenetic analysis indicated that the cDNAs represented 12 of the 14 MADS-box gene subfamilies. TaAGL14 and TaAGL15 formed a new subfamily along with a rice gene OsMADS32. RT-PCR analysis revealed that these genes had different exprsssion patterns in different organs of different stages. Expression patterns of TaAGL1 and TaAGL29 were also determined using in situ hybridization. TaAGL1 was abundantly expressed in primary root tips and the whole spikelet with more intense labeling at lodicules, paleas and stamens. TaAGL29 was expressed in both the non-reproductive parts (lemma, palea and glumes), and stamens and pistils. Moreover, differential expression patterns of these genes were also observed between wheat hybrid and its parents in leaf, stem and root of jointing stage, some were up-regulated while others were down-regulated in hybrid as compared to its parents. We concluded that multiple MADS-box genes exist in wheat genome and are expressed in tissue-specific patterns, and might play important roles in wheat growth and development.

Isolation and characterization of 18 genes encoding α- and β-expansins in wheat (Triticum aestivum L.)

Expansins are thought to be key regulators of cell wall extension during plant growth. In this study, we isolated 18 expansin genes from wheat, nine of which encode α-expansins while the other nine code for β-expansins. The cysteine-rich and tryptophan-rich regions of the deduced amino acid sequences of all 18 expansins were highly conserved. Genomic sequences were obtained for 17 of the genes, and their intron patterns were determined. Four (A, C, D, E) of the six intron positions known in expansin genes from other species were found to be occupied in these wheat expansin genes. Five wheat expansin genes were mapped to chromosomes 1L, 2L, 5L and 6L respectively, by in silico and comparative mapping. The 18 wheat expansin genes were expressed in leaf, root and the developing seed. Moreover, it was demonstrated that four β-expansin genes were up-regulated in the internode tissue in F1 hybrids, suggesting that changes in the regulation of these genes in hybrid might contribute to the heterosis observed in internode length and plant height. We therefore conclude that expansins are encoded by a multigene family in wheat, and could play important roles in growth and development.