Cloning and Expression Studies of Novel Small RNAs in Tetraploid Cotton

Small RNAs are a group of non-coding RNAs that downregulate gene expression in a sequence-specific manner to control plant growth and development. The objective of the present study was to clone and characterize several small RNAs in cotton. To identify small RNAs that are involved in the development of cotton bolls and fibers, we generated cDNA libraries from cotton bolls at 13?days post-anthesis from two cotton cultivars, Pima Phy 76 (Gossypium bardadense) and Acala 1517?C99 (Gossypium hirsutum). Screening of these libraries identified eight small RNAs, seven of which have not been reported in other plant species and appear to be absent in the known sequences of other plant species. Their predicted target genes are known to be involved in cotton fiber development. The cloned small RNAs displayed lower and differential expression in the examined boll developmental stages using RT-PCR and quantitative RT-PCR. The genetic polymorphism of the small RNAs at the DNA level was evaluated by miRNA-amplified fragment length polymorphism (AFLP) analysis using primers designed from the small miRNA genes in combination with AFLP primers. Homologous small RNA gene sequences were further isolated using this homology-based genotyping approach, and potential hairpin structures were identified. The results represent a novel method to isolate small including miRNA genes at the RNA and DNA levels in many plant species where genome sequences are not available or expressed sequence tags are limited.     

Role of MicroRNAs and small RNAs in regulation of developmental processes and agronomic traits in Gossypium species

Small RNAs (sRNAs) are short, non-coding, 17–24 nucleotides long RNA molecules that play vital roles in regulating gene expression in every known organism investigated to date including cotton (Gossypium ssp.). These tiny RNA molecules target diverse categories of genes from different bioliogical and metabolic processes and have been reported in the three domains of life. Small RNAs, including miRNAs, are involved in ovule and fiber development, biotic and abiotic stresses, fertility, and other biochemical processes in cotton species. Also, sRNAs are the critical components in RNA interference pathway. In this article, we have reviewed the research efforts related to the isolation and characterization of miRNAs using molecular and genomic approaches. The progress made in understanding the functional roles of miRNAs in regulation, alteration, and inactivation of fundamental plant processes and traits of importance in cotton are presented here.     

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。     

MicroRNA–target gene responses to lead-induced stress in cotton (Gossypium hirsutum L.)

MicroRNAs (miRNAs) play key roles in plant responses to various metal stresses. To investigate the miRNA-mediated plant response to heavy metals, cotton (Gossypium hirsutum L.), the most important fiber crop in the world, was exposed to different concentrations (0, 25, 50, 100, and 200 µM) of lead (Pb) and then the toxicological effects were investigated. The expression patterns of 16 stress-responsive miRNAs and 10 target genes were monitored in cotton leaves and roots by quantitative real-time PCR (qRT-PCR); of these selected genes, several miRNAs and their target genes are involved in root development. The results show a reciprocal regulation of cotton response to lead stress by miRNAs. The characterization of the miRNAs and the associated target genes in response to lead exposure would help in defining the potential roles of miRNAs in plant adaptation to heavy metal stress and further understanding miRNA regulation in response to abiotic stress.     

Identification and expression analysis of miRNAs from nitrogen-fixing soybean nodules

miRNAs are ～21 nt non-coding RNAs and play important roles in plant development and response to stress. Symbiotic nitrogen fixation (SNF) is agronomically important for reducing the need of nitrogen fertilizers. The soybean root nodule is the place where SNF takes place. To identify miRNAs that are possibly involved in nitrogen fixation in soybean functional nitrogen-fixing nodules, a small library of RNAs was constructed from the functional nodules harvested 28 days after inoculation with rhizobium. Thirty-two small RNA sequences were identified as belonging to 11 miRNA families. Eight miRNAs are conserved across plant species, twenty are specific to soybean, and the four remaining miRNAs are novel. Expression analysis revealed that miRNAs were differentially expressed in the different tissues. Combinatorial miRNA target prediction identified genes that are involved in multiple biological processes. The results suggest that miRNAs play critical and diverse roles in SNF, nutrient acquisition, and plant development.     

Identification and characterization of microRNAs and their target genes in Brassica oleracea

The microRNAs are a new class of small non-coding endogenous RNAs with lengths of approximately ~21 nt. MicroRNAs perform their biological function via the degradation of the target mRNAs or by inhibiting protein translation. Until recently, only limited numbers of miRNAs were identified in Brassica oleracea, a vegetable widely cultivated around the world. In present study, 193 potential miRNA candidates were identified from 17 expressed sequence tag (ESTs) and 152 genome survey sequences (GSSs) in B. oleracea. These miRNA candidates were classified into 70 families using a well-defined comparative genome-based computational analysis. Most miRNAs belong to the miRNA169, miR5021, miR156 and miR158 families. Of these, 36 miRNA families are firstly found in Brassica species. Around 1393 B. oleracea genes were predicted as candidate targets of 175 miRNAs. The mutual relationship between miRNAs and the candidate target genes was verified by checking differentially expression levels using quantitative real-time polymerase chain reaction (qRT-PCR) and 5' RLM-RACE analyses. These target genes participate in multiple biological and metabolic processes, including signal transduction, stress response, and plant development. Gene Ontology analysis shows that the 818, 514, and 265 target genes are involved in molecular functions, biological processes, and cellular component respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway enrichment analysis suggests that these miRNAs might regulate 186 metabolic pathways, including those of lipid, energy, starch and sucrose, fatty acid and nitrogen.     

Identification of new small non-coding RNAs from tobacco and Arabidopsis

Small non-coding RNAs (ncRNAs) have typically been searched in fully sequenced genomes using one of two approaches-experimental or computational. We developed a mixed method, using both types of information, which has the advantage of applying bio-computing methods to actually expressed sequences. Our method allowed the identification of new small ncRNAs in Arabidopsis thaliana and in the unfinished genome of Nicotiana tabacum. We constructed a N. tabacum cDNA library from small RNAs ranging from 20 to 30 nucleotides (nt). The sequences from 73 unique clones were compared to the A. thaliana genome and to all plant sequences using a pattern-matching approach (program Patbank). Thus, we selected 15 clones from the library corresponding mostly to A. thaliana or N. tabacum non-coding sequences. By Northern blot analyses, we confirmed the presence of most RNA candidates in Arabidopsis and in Nicotiana sylvestris with a size range of 21-100 nt. To gain more insight into the possible genesis of 21-24 nt sequences, stable folding of sRNAs with their flanking regions were predicted with the software MIRFOLD dedicated to the folding of microRNAs (miRNA). Stable hairpins structures were observed for some putative miRNAs.