Identification and characterization of salt-responsive microRNAs in Populus tomentosa by high-throughput sequencing

Salt is one of the main environmental factors limiting plant growth and a better understanding of mechanisms of salt stress would aid efforts to bolster plant salt tolerance. MicroRNAs are well known for their important regulatory roles in response to abiotic stress in plants. In this study, high-throughput sequencing was employed to identify miRNAs in Populus tomentosa plantlets treated or not with salt (200 mM for 10 h). We found 141 conserved miRNAs belonging to 31 families, 29 non-conserved but previously-known miRNAs belonging to 26 families, and 17 novel miRNAs. Under salt stress, 19 miRNAs belonging to seven conserved miRNA families were significantly downregulated, and two miRNAs belonging to two conserved miRNA families were upregulated. Of seven non-conserved miRNAs with significantly altered expression, five were downregulated and two were upregulated. Furthermore, eight miRNAs were validated by qRT-PCR and their dynamic differential expressions were analyzed. In addition, 269 target genes of identified miRNAs were predicted and categorized by function. These results provide new insights into salt-responsive miRNAs in Populus.     

High-throughput sequence analysis of small RNAs in skotomorphogenic seedlings of Brassica rapa ssp. rapa

Skotomorphogenic development is the process by which seedlings adapt to a stressful dark environment. Such metabolic responses to abiotic stresses in plants are known to be regulated in part by microRNAs (miRNAs); however, little is known about the involvement of miRNAs in the regulation of skotomorphogenesis. To identify miRNAs at the genome-wide level in skotomorphogenic seedlings of turnip (Brassica rapa subsp. rapa), an important worldwide root vegetable, we used Solexa sequencing to sequence a small RNA library from seedlings grown in the dark for 4 days. Deep sequencing showed that the small RNAs (sRNAs) were predominantly 21 to 24 nucleotides long. Specifically, 13,319,035 reads produced 359,531 unique sRNAs including rRNA, tRNA, miRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and unannotated sRNAs. Sequence analysis identified 96 conserved miRNAs belonging to 36 miRNA families and 576 novel miRNAs. qRT-PCR confirmed that the miRNAs were expressed during skotomorphogenesis similar to the trends shown by the Solexa sequencing results. A total of 2013 potential targets were predicted, and the targets of BrmiR157, BrmiR159 and BrmiR160 were proved to be regulated by miRNA-guided cleavage. These results show that specific regulatory miRNAs are present in skotomorphogenic seedlings of turnip and may play important roles in growth, development, and response to dark environment.     

Genome-wide profiling of novel and conserved <Emphasis Type="Italic">Populus</Emphasis> microRNAs involved in pathogen stress response by deep sequencing

MicroRNAs (miRNAs) are small RNAs, generally of 20–23 nt, that down-regulate target gene expression during development, differentiation, growth, and metabolism. In Populus, extensive studies of miRNAs involved in cold, heat, dehydration, salinity, and mechanical stresses have been performed; however, there are few reports profiling the miRNA expression patterns during pathogen stress. We obtained almost 38 million raw reads through Solexa sequencing of two libraries from Populus inoculated and uninoculated with canker disease pathogen. Sequence analyses identified 74 conserved miRNA sequences belonging to 37 miRNA families from 154 loci in the Populus genome and 27 novel miRNA sequences from 35 loci, including their complementary miRNA* strands. Intriguingly, the miRNA* of three conserved miRNAs were more abundant than their corresponding miRNAs. The overall expression levels of conserved miRNAs increased when subjected to pathogen stress, and expression levels of 33 miRNA sequences markedly changed. The expression trends determined by sequencing and by qRT-PCR were similar. Finally, nine target genes for three conserved miRNAs and 63 target genes for novel miRNAs were predicted using computational analysis, and their functions were annotated. Deep sequencing provides an opportunity to identify pathogen-regulated miRNAs in trees, which will help in understanding the regulatory mechanisms of plant defense responses during pathogen infection.     

Discovery and profiling of novel and conserved microRNAs during flower development in Carya cathayensis via deep sequencing

Hickory (Carya cathayensis Sarg.) is an economically important woody plant in China, but its long juvenile phase delays yield. MicroRNAs (miRNAs) are critical regulators of genes and important for normal plant development and physiology, including flower development. We used Solexa technology to sequence two small RNA libraries from two floral differentiation stages in hickory to identify miRNAs related to flower development. We identified 39 conserved miRNA sequences from 114 loci belonging to 23 families as well as two novel and ten potential novel miRNAs belonging to nine families. Moreover, 35 conserved miRNA*s and two novel miRNA*s were detected. Twenty miRNA sequences from 49 loci belonging to 11 families were differentially expressed; all were up-regulated at the later stage of flower development in hickory. Quantitative real-time PCR of 12 conserved miRNA sequences, five novel miRNA families, and two novel miRNA*s validated that all were expressed during hickory flower development, and the expression patterns were similar to those detected with Solexa sequencing. Finally, a total of 146 targets of the novel and conserved miRNAs were predicted. This study identified a diverse set of miRNAs that were closely related to hickory flower development and that could help in plant floral induction.     

植物microRNA的保守性及其分布的分析

MicroRNA(miRNA)是真核生物中具有重要调控作用的小分子非编码RNA。本文对miRNA官网miRBase数据库Release 22.1中隶属于植物界的绿藻门、苔藓植物门、蕨类植物门、裸子植物门、被子植物门共计82个物种的miRNA进行了统计分析。miRBase共收录植物miRNA 前体8 615个,成熟miRNA 10 414条,隶属于2 892个miRNA家族。绿藻门miRNA与其他4个门miRNA无同源性;对其他4个门植物miRNA的保守性进行研究,发现存在于2个植物门的miRNA家族有26个,属于中度保守miRNA家族;14个miRNA家族存在于3个及3个以上植物门中,属于高度保守miRNA家族,其中7个miRNA家族系苔藓、蕨类、裸子和被子植物共有,是植物中最保守的miRNA。分析表明,超过30个miRNA家族的植物有35种。进一步对40个中度或者高度保守miRNA在35种植物中的分布进行研究,发现miRNA家族及其成员在物种间的分布存在较大的差异。这些分布上的差异一方面反映不同植物中miRNA的研究深度不同,另一方面也反映出miRNA在植物进化过程中的适应性调整。研究不同植物中miRNA家族的分布,可在miRNA水平为植物早期进化同源性的研究提供分子依据。     

植物microRNA的保守性及其分布的分析

MicroRNA(miRNA)是真核生物中具有重要调控作用的小分子非编码RNA。本文对miRNA官网miRBase数据库Release 22.1中隶属于植物界的绿藻门、苔藓植物门、蕨类植物门、裸子植物门、被子植物门共计82个物种的miRNA进行了统计分析。miRBase共收录植物miRNA 前体8 615个,成熟miRNA 10 414条,隶属于2 892个miRNA家族。绿藻门miRNA与其他4个门miRNA无同源性;对其他4个门植物miRNA的保守性进行研究,发现存在于2个植物门的miRNA家族有26个,属于中度保守miRNA家族;14个miRNA家族存在于3个及3个以上植物门中,属于高度保守miRNA家族,其中7个miRNA家族系苔藓、蕨类、裸子和被子植物共有,是植物中最保守的miRNA。分析表明,超过30个miRNA家族的植物有35种。进一步对40个中度或者高度保守miRNA在35种植物中的分布进行研究,发现miRNA家族及其成员在物种间的分布存在较大的差异。这些分布上的差异一方面反映不同植物中miRNA的研究深度不同,另一方面也反映出miRNA在植物进化过程中的适应性调整。研究不同植物中miRNA家族的分布,可在miRNA水平为植物早期进化同源性的研究提供分子依据。     

Genome-wide identification of cold-responsive and new microRNAs in Populus tomentosa by high-throughput sequencing

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate the expression of target mRNAs in plant growth, development, abiotic stress responses, and pathogen responses. Cold stress is one of the most common abiotic factors affecting plants, and it adversely affects plant growth, development, and spatial distribution. To understand the roles of miRNAs under cold stress in Populus tomentosa, we constructed two small RNA libraries from plantlets treated or not with cold conditions (4 °C for 8 h). High-throughput sequencing of the two libraries identified 144 conserved miRNAs belonging to 33 miRNA families and 29 new miRNAs (as well as their corresponding miRNA¹s) belonging to 23 miRNA families. Differential expression analysis showed that 21 miRNAs were down-regulated and nine miRNAs were up-regulated in response to cold stress. Among them, 19 cold-responsive miRNAs, two new miRNAs and their corresponding miRNA¹s were validated by qRT-PCR. A total of 101 target genes of the new miRNAs were predicted using a bioinformatics approach. These target genes are involved in growth and resistance to various stresses. The results demonstrated that Populus miRNAs play critical roles in the cold stress response.     

Identification of microRNAs and their targets from Populus euphratica

Populus euphratica is an ideal model system for research into the abiotic stress resistance research of woody plants. Although microRNAs have been found in poplars and have been shown to have diverse biological functions, a majority of them are genus- or specie-specific and few microRNAs have been found in P. euphratica to date. In this study, microRNA cloning and computational expressed sequence tag analysis were used to identify 72 putative miRNA sequences in P. euphratica. These sequences could be classified into 21 families, 12 of which were novel, increasing the number of known poplar microRNA families from 42 to 54. Expression analysis indicated that five P. euphratica microRNAs were induced by dehydration stress. Bioinformatics prediction showed that the 130 target genes are involved in development, resistance to stress, and other cellular processes. These results suggest several roles for miRNAs in the regulatory networks associated with the abiotic stress resistance of tree species.     

Identification and characterisation of microRNAs in young adults of Angiostrongylus cantonensis via a deep-sequencing approach

Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. MicroRNAs (miRNAs) are small non-coding RNAs that participate in a wide range of biological processes. This study employed a deep-sequencing approach to study miRNAs from young adults of A. cantonensis. Based on 16,880,456 high-quality reads, 252 conserved mature miRNAs including 10 antisense miRNAs that belonging to 90 families, together with 10 antisense miRNAs were identified and characterised. Among these sequences, 53 miRNAs from 25 families displayed 50 or more reads. The conserved miRNA families were divided into four groups according to their phylogenetic distribution and a total of nine families without any members showing homology to other nematodes or adult worms were identified. Stem-loop real-time polymerase chain reaction analysis of aca-miR-1-1 and aca-miR-71-1 demonstrated that their level of expression increased dramatically from infective larvae to young adults and then decreased in adult worms, with the male worms exhibiting significantly higher levels of expression than female worms. These findings provide information related to the regulation of gene expression during the growth, development and pathogenesis of young adults of A. cantonensis.