Identification of the Conserved and Novel miRNAs in Mulberry by High-Throughput Sequencing

miRNAs are a class of non-coding endogenous small RNAs. They play vital roles in plant growth, development, and response to biotic and abiotic stress by negatively regulating genes. Mulberry trees are economically important species with multiple uses. However, to date, little is known about mulberry miRNAs and their target genes. In the present study, three small mulberry RNA libraries were constructed and sequenced using high-throughput sequencing technology. Results showed 85 conserved miRNAs belonging to 31 miRNA families and 262 novel miRNAs at 371 loci. Quantitative real-time PCR (qRT-PCR) analysis confirmed the expression pattern of 9 conserved and 5 novel miRNAs in leaves, bark, and male flowers. A total of 332 potential target genes were predicted to be associated with these 113 novel miRNAs. These results provide a basis for further understanding of mulberry miRNAs and the biological processes in which they are involved.     

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.     

蒙古沙冬青保守microRNAs的鉴定及靶基因预测

蒙古沙冬青(Ammopiptanthus mongolicus)是生长在荒漠中的木本植物, 对于我国西北部干旱、半干旱区域的植被维护与恢复具有重要价值。蒙古沙冬青对干旱、低温等多种逆境具有较高的耐受性, 是研究林木耐受逆境生理与分子机制的合适材料。MicroRNA(miRNA)是一类长度约为21个核苷酸的内源性非编码小分子RNA, 在植物生长发育和逆境应答等生物学过程中发挥着重要的调控作用。目前, 许多植物物种的miRNAs已经获得鉴定, 但未见蒙古沙冬青miRNAs的相关报道。文章应用高通量测序和生物信息学分析方法对蒙古沙冬青幼苗保守miRNAs的类型、表达丰度以及靶基因进行了分析和预测。共鉴定了10个家族的19种保守miRNAs, 其表达丰度介于55~1920269个拷贝之间。通过在线软件psRNATarget预测了其中14个保守miRNAs的靶基因。对于这些靶基因的功能分析表明, 蒙古沙冬青的保守miRNA主要通过转录调控、激素信号途径、物质代谢和胁迫应答等生物学过程参与植物生长发育和环境响应。     

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.     

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.     

Analyses of a Glycine max Degradome Library Identify microRNA Targets and MicroRNAs that Trigger Secondary SiRNA Biogenesis

Plant microRNAs (miRNAs) regulate gene expression mainly by guiding cleavage of target mRNAs. In this study, a degradome library constructed from different soybean (Glycine max (L.) Merr.) tissues was deep-sequenced. 428 potential targets of small interfering RNAs and 25 novel miRNA families were identified. A total of 211 potential miRNA targets, including 174 conserved miRNA targets and 37 soybean-specific miRNA targets, were identified. Among them, 121 targets were first discovered in soybean. The signature distribution of soybean primary miRNAs (pri-miRNAs) showed that most pri-miRNAs had the characteristic pattern of Dicer processing. The biogenesis of TAS3 small interfering RNAs (siRNAs) was conserved in soybean, and nine Auxin Response Factors were identified as TAS3 siRNA targets. Twenty-three miRNA targets produced secondary small interfering RNAs (siRNAs) in soybean. These targets were guided by five miRNAs: gma-miR393, gma-miR1508, gma-miR1510, gma-miR1514, and novel-11. Multiple targets of these secondary siRNAs were detected. These 23 miRNA targets may be the putative novel TAS genes in soybean. Global identification of miRNA targets and potential novel TAS genes will contribute to research on the functions of miRNAs in soybean.     

miR398在植物逆境胁迫应答中的作用

MicroRNA (miRNA)是一类新型的调控基因表达的小分子RNA, 它作为基因表达的负调控因子, 在转录后水平调节靶基因的表达。miRNA参与调控植物的生长发育, 并在多种非生物与生物胁迫响应中发挥重要作用。miR398是第一个被报道的受氧化胁迫负调控的miRNA。它通过负调控其靶基因Cu/Zn过氧化物歧化酶(Cu/Zn-superoxide dismutase, CSD)的表达, 在多种逆境胁迫响应中扮演重要角色, 如调节铜代谢平衡, 应答重金属、蔗糖、臭氧等非生物胁迫, 以及参与应答生物胁迫等。文章综述了miR398在多种逆境胁迫响应中重要的调节作用及miR398自身的转录调控。     

Computational identification of novel family members of microRNA genes in Arabidopsis thaliana and Oryza sativa

MicroRNAs (miRNAs) are a class of endogenous small RNAs that play important regulatory roles in both animals and plants, miRNA genes have been intensively studied in animals, but not in plants. In this study, we adopted a homology search approach to identify homologs of previously validated plant miRNAs in Arabidopsis thaliana and Oryza sativa. We identified 20 potential miRNA genes in Arabidopsis and 40 in O. sativa, providing a relatively complete enumeration of family members for these miRNAs in plants. In addition, a greater number of Arabidopsis miRNAs (MIR168, MIR159 and MIR172) were found to be conserved in rice. With the novel homologs, most of the miRNAs have closely related fellow miRNAs and the number of paralogs varies in the different miRNA families. Moreover, a probable functional segment highly conserved on the elongated stem of pre-miRNA fold-backs of MIR319 and MIR159 family was identified. These results support a model of variegated miRNA regulation in plants, in which miRNAs with different functional elements on their pre-miRNA fold-backs can differ in their function or regulation, and closely related miRNAs can be diverse in their specificity or competence to downregulate target genes. It appears that the sophisticated regulation of miRNAs can achieve complex biological effects through qualitative and quantitative modulation of gene expression profiles in plants.