利用深度测序技术检测玉米根系和叶片中已知的microRNAs

microRNA(miRNA)是一类具有20～24nt核苷酸长度的非蛋白质编码的内源小分子RNA,它在植物生长发育和逆境胁迫响应等过程中发挥着重要作用。文章利用基于Illumina/Solexa原理的小分子RNA深度测序技术,结合生物信息学的方法对玉米根系和叶片中已知miRNA的类型、丰度及靶基因进行了分析。研究发现,在根系中共检测到92个已知的miRNA,分别属于18个miRNA家族,其表达丰度在1～105943之间;在叶片中,共发现86个已知的miRNA,分别属于17个miRNA家族,其表达丰度在1～85973之间。靶基因预测结果表明,根系中的18个miRNA家族共靶向54个蛋白,进一步的功能预测发现,这些基因涉及了转录调控、物质能量代谢、电子传递、胁迫响应和信号转导等过程。以上研究结果表明,就已知的miRNA而言,无论是miRNA的类型还是表达丰度,在玉米根系和叶片中都存在较大差异。     

Genome‐wide identification and characterization of microRNAs responding to ABA and GA in maize embryos during seed germination

• MicroRNAs (miRNAs) are an important class of non‐coding small RNAs that regulate the expression of target genes through mRNA cleavage or translational inhibition. Previous studies have revealed their roles in regulating seed dormancy and germination in model plants such as Arabidopsis thaliana, rice (Oryza sativa) and maize (Zea mays). However, the miRNA response to exogenous gibberellic acid (GA) and abscisic acid (ABA) during seed germination in maize has yet to be explored.
• In this study, small RNA libraries were generated and sequenced from maize embryos treated with GA, ABA or double‐distilled water as control.
• A total of 247 miRNAs (104 known and 143 novel) were identified, of which 45 known and 53 novel miRNAs were differentially expressed in embryos in the different treatment groups. In total, 74 (37 up‐regulated and 37 down‐regulated) and 55 (23 up‐regulated and 32 down‐regulated) miRNAs were expressed in response to GA and to ABA, respectively, and a total of 18 known and 38 novel miRNAs displayed differential expression between the GA‐ and ABA‐treated groups. Using bioinformatics tools, we predicted the target genes of the differentially expressed miRNAs. Using GO enrichment and KEGG pathway analysis of these targets, we showed that miRNAs differentially expressed in our samples affect genes encoding proteins involved in the peroxisome, ribosome and plant hormonal signalling pathways.
• Our results indicate that miRNA‐mediated gene expression influences the GA and ABA signalling pathways during seed germination.

     

System analysis of microRNAs in the development and aluminium stress responses of the maize root system

MicroRNAs (miRNAs) are a class of regulatory small RNAs (sRNAs) that down‐regulate target genes through mRNA cleavage or translational inhibition. miRNA is known to play an important role in the root development and environmental responses in both the Arabidopsis and rice. However, little information is available to form a complete view of miRNAs in the development of the maize root system and Al stress responses in maize. Four sRNA libraries were generated and sequenced from the early developmental stage of primary roots (PRY), the later developmental stage of maize primary roots (PRO), seminal roots (SR) and crown roots (CR). Through integrative analysis, we identified 278 miRNAs (246 conserved and 32 novel ones) and found that the expression patterns of miRNAs differed dramatically in different maize roots. The potential targets of the identified conserved and novel miRNAs were also predicted. In addition, our data showed that CR is more resistant to Al stress compared with PR and SR, and the differentially expressed miRNAs are likely to play significant roles in different roots in response to environmental stress such as Al stress. Here, we demonstrate that the expression patterns of miRNAs are highly diversified in different maize roots. The differentially expressed miRNAs are correlated with both the development and environmental responses in the maize root. This study not only improves our knowledge about the roles of miRNAs in maize root development but also reveals the potential role of miRNAs in the environmental responses of different maize roots.     

Cloning and characterization of miRNAs from maize seedling roots under low phosphorus stress

MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition in plants and animals. In this study, a small RNA library was constructed to identify conserved miRNAs as well as novel miRNAs in maize seedling roots under low level phosphorus stress. Twelve miRNAs were identified by high throughput sequencing of the library and subsequent analysis, two belong to conserved miRNA families (miRNA399b and miRNA156), and the remaining ten are novel and one of latter is conserved in gramineous species. Based on sequence homology, we predicted 125 potential target genes of these miRNAs and then expression patterns of 7 miRNAs were validated by semi-RT-PCR analysis. MiRNA399b, Zma-miR3, and their target genes (Zmpt1 and Zmpt2) were analyzed by real-time PCR. It is shown that both miRNA399b and Zma-miR3 are induced by low phosphorus stress and regulated by their target genes (Zmpt1 and Zmpt2). Moreover, Zma-miR3, regulated by two maize inorganic phosphate transporters as a newly identified miRNAs, would likely be directly involved in phosphate homeostasis, so was miRNA399b in Arabidopsis and rice. These results indicate that both conserved and maize-specific miRNAs play important roles in stress responses and other physiological processes correlated with phosphate starvation, regulated by their target genes. Identification of these differentially expressed miRNAs will facilitate us to uncover the molecular mechanisms underlying the progression of maize seedling roots development under low level phosphorus stress.     

玉米microRNAs及其靶基因的生物信息学预测

microRNAs (miRNAs) 是一类非编码的小分子RNA, 通过碱基互补调控靶基因的表达。鉴定和发现新的miRNAs及其靶基因, 对揭示miRNAs在基因表达调控中的作用至关重要。玉米全基因组测序工作开展较晚, 已经鉴定登记的miRNAs很少, 对靶基因的调控作用尚待解明。文章根据miRNA进化上的保守性, 以已知的植物miRNAs为探针, 与相关数据库中玉米表达序列标签(EST)和基因组序列(GSS)中的非编码序列比对, 共发现11个新的miRNA前体。虽然在序列长度和二级结构方面各有变化, 但这11个前体均可折叠形成miRNA家族的标准二级结构。通过靶基因预测, 找到其中7条miRNAs的26个靶基因, 分别编码与新陈代谢、信号转导、转录调节、跨膜运输、生物和非生物胁迫及叶绿体组装等相关的蛋白。这些miRNAs及其靶基因的鉴定, 补充了miRNA数据库的不足。     

Analysis of physiological and miRNA responses to Pi deficiency in alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.)

Key message

The induction of miR399 and miR398 and the inhibition of miR156, miR159, miR160, miR171, miR2111, and miR2643 were observed under Pi deficiency in alfalfa. The miRNA-mediated genes involved in basic metabolic process, root and shoot development, stress response and Pi uptake.

Abstract

Inorganic phosphate (Pi) deficiency is known to be a limiting factor in plant development and growth. However, the underlying miRNAs associated with the Pi deficiency-responsive mechanism in alfalfa are unclear. To elucidate the molecular mechanism at the miRNA level, we constructed four small RNA (sRNA) libraries from the roots and shoots of alfalfa grown under normal or Pi-deficient conditions. In the present study, alfalfa plants showed reductions in biomass, photosynthesis, and Pi content and increases in their root-to-shoot ratio and citric, malic, and succinic acid contents under Pi limitation. Sequencing results identified 47 and 44 differentially expressed miRNAs in the roots and shoots, respectively. Furthermore, 909 potential target genes were predicted, and some targets were validated by RLM-RACE assays. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed prominent enrichment in signal transducer activity, binding and basic metabolic pathways for carbohydrates, fatty acids and amino acids; cellular response to hormone stimulus and response to auxin pathways were also enriched. qPCR results verified that the differentially expressed miRNA profile was consistent with sequencing results, and putative target genes exhibited opposite expression patterns. In this study, the miRNAs associated with the response to Pi limitation in alfalfa were identified. In addition, there was an enrichment of miRNA-targeted genes involved in biological regulatory processes such as basic metabolic pathways, root and shoot development, stress response, Pi transportation and citric acid secretion.

     

SIZ1 regulation of phosphate starvation-induced root architecture remodeling involves the control of auxin accumulation

Phosphate (Pi) limitation causes plants to modulate the architecture of their root systems to facilitate the acquisition of Pi. Previously, we reported that the Arabidopsis (Arabidopsis thaliana) SUMO E3 ligase SIZ1 regulates root architecture remodeling in response to Pi limitation; namely, the siz1 mutations cause the inhibition of primary root (PR) elongation and the promotion of lateral root (LR) formation. Here, we present evidence that SIZ1 is involved in the negative regulation of auxin patterning to modulate root system architecture in response to Pi starvation. The siz1 mutations caused greater PR growth inhibition and LR development of seedlings in response to Pi limitation. Similar root phenotypes occurred if Pi-deficient wild-type seedlings were supplemented with auxin. N-1-Naphthylphthalamic acid, an inhibitor of auxin efflux activity, reduced the Pi starvation-induced LR root formation of siz1 seedlings to a level equivalent to that seen in the wild type. Monitoring of the auxin-responsive reporter DR5::uidA indicated that auxin accumulates in PR tips at early stages of the Pi starvation response. Subsequently, DR5::uidA expression was observed in the LR primordia, which was associated with LR elongation. The time-sequential patterning of DR5::uidA expression occurred earlier in the roots of siz1 as compared with the wild type. In addition, microarray analysis revealed that several other auxin-responsive genes, including genes involved in cell wall loosening and biosynthesis, were up-regulated in siz1 relative to wild-type seedlings in response to Pi starvation. Together, these results suggest that SIZ1 negatively regulates Pi starvation-induced root architecture remodeling through the control of auxin patterning.     

Characterization and Expression Patterns of microRNAs Involved in Rice Grain Filling

MicroRNAs (miRNAs) are upstream gene regulators of plant development and hormone homeostasis through their directed cleavage or translational repression of the target mRNAs, which may play crucial roles in rice grain filling and determining the final grain weight and yield. In this study, high-throughput sequencing was performed to survey the dynamic expressions of miRNAs and their corresponding target genes at five distinct developmental stages of grain filling. In total, 445 known miRNAs and 45 novel miRNAs were detected with most of them expressed in a developmental stage dependent manner, and the majority of known miRNAs, which increased gradually with rice grain filling, showed negatively related to the grain filling rate. Detailed expressional comparisons revealed a clear negative correlation between most miRNAs and their target genes. It was found that specific miRNA cohorts are expressed in a developmental stage dependent manner during grain filling and the known functions of these miRNAs are involved in plant hormone homeostasis and starch accumulation, indicating that the expression dynamics of these miRNAs might play key roles in regulating rice grain filling.     

Comparison of microRNAs in adipose and muscle tissue from seven indigenous Chinese breeds and Yorkshire pigs

Elucidation of the pig microRNAome is essential for interpreting functional elements of the genome and understanding the genetic architecture of complex traits. Here, we extracted small RNAs from skeletal muscle and adipose tissue, and we compared their expression levels between one Western breed (Yorkshire) and seven indigenous Chinese breeds. We detected the expression of 172 known porcine microRNAs (miRNAs) and 181 novel miRNAs. Differential expression analysis found 92 and 12 differentially expressed miRNAs in adipose and muscle tissue respectively. We found that different Chinese breeds shared common directional miRNA expression changes compared to Yorkshire pigs. Some miRNAs differentially expressed across multiple Chinese breeds, including ssc‐miR‐129‐5p, ssc‐miR‐30 and ssc‐miR‐150, are involved in adipose tissue function. Functional enrichment analysis revealed that the target genes of the differentially expressed miRNAs are associated mainly with signaling pathways rather than metabolic and biosynthetic processes. The miRNA–target gene and miRNA–phenotypic traits networks identified many hub miRNAs that regulate a large number of target genes or phenotypic traits. Specifically, we found that intramuscular fat content is regulated by the greatest number of miRNAs in muscle tissue. This study provides valuable new candidate miRNAs that will aid in the improvement of meat quality and production.