MiR319-targeted OsTCP21 and OsGAmyb regulate tillering and grain yield in rice

Multiple genes and microRNAs (miRNAs) improve grain yield by promoting tillering. MiR319s are known to regulate several aspects of plant development; however, whether miR319s are essential for tillering regulation remains unclear. Here, we report that miR319 is highly expressed in the basal part of rice plant at different development stages. The miR319 knockdown line Short Tandem Target Mimic 319 (STTM319) showed higher tiller bud length in seedlings under low nitrogen (N) condition and higher tiller bud number under high N condition compared with the miR319a-overexpression line. Through targets prediction, we identified OsTCP21 and OsGAmyb as downstream targets of miR319. Moreover, OsTCP21 and OsGAmyb overexpression lines and STTM319 had increased tiller bud length and biomass, whereas both were decreased in OsTCP21 and OsGAmyb knockout lines and OE319a. These data suggest that miR319 regulates rice tiller bud development and tillering through targeting OsTCP21 and OsGAmyb. Notably, the tiller number and grain yield increased in STTM319 and overexpression lines of OsTCP21 and OsGAmyb but decreased in OE319a and knockout lines of OsTCP21 and OsGAmyb. Taken together, our findings indicate that miR319s negatively affect tiller number and grain yield by targeting OsTCP21 and OsGAmyb, revealing a novel function for miR319 in rice.     

盐胁迫下大豆根组织定量PCR分析中内参基因的选择

实时荧光定量PCR已广泛用于基因表达的分析, 适当的内参基因选择是获得准确分析结果的关键。在大豆(Glycine max)分子生物学研究中, 逆境响应基因和microRNA (miRNA)表达的内参辅助检测基因均有哪些目前尚不清楚。该研究选用不同盐梯度和时间点组合处理的大豆根组织为材料, 对已报道的其它条件下表达相对稳定的内参基因(ACT、ACT2/7、CYP2、ELF1A、ELF1B、F-Box、TUA和UBC2)以及miRNA内参基因(U6、miR1515a、miR1520c、miR1520d、miR171a和miR171b)的表达情况进行了全面检测; 并采用Δ-Ct、Bestkeeper、NormFinder和Genorm四种方法对检测结果进行了综合分析, 发现ELF1B和CYP2适合作为大豆根系盐胁迫响应基因研究的内参基因, miR1515a和U6适合作为盐胁迫下大豆根组织miRNA研究的内参。上述研究结果为大豆盐胁迫响应基因和miRNA表达及其进一步的功能研究奠定了基础。     

MIR166基因家族在陆生植物中的进化模式分析

MicroRNA（miRNA）是一类广泛存在于真核生物中的具有转录后水平调控功能的内源非编码小分子RNA。在植物中．miRNA通过对靶基因的剪切或沉默来实现对植物生命活动的调控,它是基因表达调控网络的重要组成部分。miR165／166（miR166）是陆生植物中最为古老的MIRNA家族之一,它通过对3型同源异域型-亮氨酸拉链（1id—ZIPⅢ）等靶标的调控,在植物的众多发育时期起着关键的调控作用。本文分析了MIR166基因在陆生植物中的进化关系,并对MIR166在基部陆生植物小立碗藓（Physcomitrella patens）中的复制及进化进行了研究。此外,HD—ZIPⅢ蛋白是植物中重要的一类转录因子,miR166对HD-ZIP Ⅲ基因的调控作用在陆地植物保守的存在,本文对HD—ZIP Ⅲ基因和miR166在进化中的相互作用进行了初步的探讨。     

Soybean AP1 homologs control flowering time and plant height

Flowering time and plant height are key agronomic traits that directly affect soybean (Glycine max) yield. APETALA1 (AP1) functions as a class A gene in the ABCE model for floral organ development, helping to specify carpel, stamen, petal, and sepal identities. There are four AP1 homologs in soybean, all of which are mainly expressed in the shoot apex. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR) – CRISPR‐associated protein 9 technology to generate a homozygous quadruple mutant, gmap1, with loss‐of‐function mutations in all four GmAP1 genes. Under short‐day (SD) conditions, the gmap1 quadruple mutant exhibited delayed flowering, changes in flower morphology, and increased node number and internode length, resulting in plants that were taller than the wild type. Conversely, overexpression of GmAP1a resulted in early flowering and reduced plant height compared to the wild type under SD conditions. The gmap1 mutant and the overexpression lines also exhibited altered expression of several genes related to flowering and gibberellic acid metabolism, thereby providing insight into the role of GmAP1 in the regulatory networks controlling flowering time and plant height in soybean. Increased node number is the trait with the most promise for enhancing soybean pod number and grain yield. Therefore, the mutant alleles of the four AP1 homologs described here will be invaluable for molecular breeding of improved soybean yield.     

SmGGPPS2对丹参酮合成的影响

香叶基香叶基焦磷酸合酶(GGPPS)是植物二萜类次生代谢物合成过程中的重要调控位点。在药用模式植物丹参(Salvia miltiorrhiza)中, GGPPS基因家族成员SmGGPPS2的生物学功能及其在丹参酮有效成分合成过程中的作用尚不明确。分别在丹参植株中过表达和RNA干涉SmGGPPS2基因, 并对转基因丹参中丹参酮含量和丹参酮合成相关基因表达量 以及转基因植物生理指标进行检测, 结果表明, 过表达SmGGPPS2株系中的丹参酮IIA和铁锈醇等脂溶性成分含量高于野生型; RNA干涉SmGGPPS2株系中的丹参酮IIA和铁锈醇等脂溶性成分含量均低于野生型。调节表达SmGGPPS2后, 丹参株系中SmHMGR1和SmCPS1等多个关键酶基因的表达都呈现明显的变化。此外, 调节表达SmGGPPS2还影响丹参植株抗性。以上结果表明, SmGGPPS2在丹参酮等萜类物质的合成中起重要的调控作用。     

The Sl TPL3–Sl WUS module regulates multi-locule formation in tomato by modulating auxin and gibberellin levels in the shoot apical meristem

Malformed fruits depreciate a plant’s market value.In tomato(Solanum lycopersicum),fruit malformation is associated with the multi-locule trait,which involves genes regulating shoot apical meristem(SAM) development.The expression pattern of TOPLESS3(SITPL3) throughout SAM development prompted us to investigate its functional significance via RNA interference(RNAi) and clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9(Cas9)-mediated gene editing.Lower SITPL3 t...     

胡杨无性系幼苗响应盐胁迫的miRNA表达差异研究

植物miRNA在调控基因表达、细胞周期、生物体发育、抗逆等方面起重要作用。为研究胡杨(Populus euphratica Oliv.)的耐盐机制,以1年生胡杨无性系幼苗为材料,构建具有空间代表性的盐胁迫胡杨cDNA文库,利用二代测序技术测定NaCl胁迫下和正常培养条件下胡杨叶和根miRNA表达情况。结果表明,不同的miRNA之间表达量存在明显差异,表达丰度最高的miRNA有miR156、miR157、miR165、miR166和miR167等,合计占总表达量的90%以上。胡杨根部存在特异表达的miRNA,在整个耐盐调控机制中发挥着生理调节、分子调控和信号传导等极为重要的作用。盐处理样品中发现大量响应盐胁迫的miRNA,对这些转录因子进行靶基因预测和注释后,发现很多盐胁迫响应的miRNA与NAC和SPL等重要转录因子家族相关,与前人的结论一致,另外还发现许多miRNA的调控对象是ATP酶和激素响应因子。     

The lncRNA39896–miR166b–HDZs module affects tomato resistance to Phytophthora infestans

The yield and quality of tomatoes(Solanum lycopersicum) is seriously affected by Phytophthora infestans. The long non-coding RNA(lnc RNA)Sl-lnc RNA39896 is induced after P. infestans infection and was previously predicted to act as an endogenous target mimic(eTM) for the micro RNA Sl-miR166b, which function in stress responses. Here, we further examined the role of Sl-lncRNA39896 and Sl-mi R166b in tomato resistance to P. infestans. Sl-miR166b levels were higher in Sl-lnc RNA39896-knockout mutan...     

Roles of small RNAs in soybean defense against Phytophthora sojae infection

The genus Phytophthora consists of many notorious pathogens of crops and forestry trees. At present, battling Phytophthora diseases is challenging due to a lack of understanding of their pathogenesis. We investigated the role of small RNAs in regulating soybean defense in response to infection by Phytophthora sojae, the second most destructive pathogen of soybean. Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are universal regulators that repress target gene expression in eukaryotes. We identified known and novel small RNAs that differentially accumulated during P. sojae infection in soybean roots. Among them, miR393 and miR166 were induced by heat‐inactivated P. sojae hyphae, indicating that they may be involved in soybean basal defense. Indeed, knocking down the level of mature miR393 led to enhanced susceptibility of soybean to P. sojae; furthermore, the expression of isoflavonoid biosynthetic genes was drastically reduced in miR393 knockdown roots. These data suggest that miR393 promotes soybean defense against P. sojae. In addition to miRNAs, P. sojae infection also resulted in increased accumulation of phased siRNAs (phasiRNAs) that are predominantly generated from canonical resistance genes encoding nucleotide binding‐leucine rich repeat proteins and genes encoding pentatricopeptide repeat‐containing proteins. This work identifies specific miRNAs and phasiRNAs that regulate defense‐associated genes in soybean during Phytophthora infection.     

Bacillus amyloliquefaciens FZB42 represses plant miR846 to induce systemic resistance via a jasmonic acid‐dependent signalling pathway

Bacillus amyloliquefaciens FZB42 is a type of plant growth‐promoting rhizobacterium (PGPR) which activates induced systemic resistance (ISR) in Arabidopsis. Blocking of the synthesis of cyclic lipopeptides and 2,3‐butanediol by FZB42, which have been demonstrated to be involved in the priming of ISR, results in the abolishment of the plant defence responses. To further clarify the ISR activated by PGPRs at the microRNA (miRNA) level, small RNA (sRNA) libraries from Arabidopsis leaves after root irrigation with FZB42, FZB42ΔsfpΔalsS and control were constructed and sequenced. After fold change selection, promoter analysis and target prediction, miR846‐5p and miR846‐3p from the same precursor were selected as candidate ISR‐associated miRNAs. miR846 belongs to the non‐conserved miRNAs, specifically exists in Arabidopsis and its function in the plant defence response remains unclear. The disease severity of transgenic Arabidopsis overexpressing miR846 (OEmiR846) or knockdown miR846 (STTM846) against Pseudomonas syringae DC3000 suggests that the miR846 expression level in Arabidopsis is negatively correlated with disease resistance. Moreover, miR846 in Arabidopsis Col‐0 is repressed after methyl jasmonate treatment. In addition, jasmonic acid (JA) signalling‐related genes are up‐regulated in STTM846, and the stomatal apertures of STTM846 are also less than those in Arabidopsis Col‐0 after methyl jasmonate treatment. Furthermore, the disease resistance of STTM846 transgenic Arabidopsis against Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is blocked by the addition of the JA biosynthetic inhibitor diethyldiethiocarbamic acid (DIECA). Taken together, our results suggest that B. amyloliquefaciens FZB42 inoculation suppresses miR846 expression to induce Arabidopsis systemic resistance via a JA‐dependent signalling pathway.     

In silico Characterisation and Phylogenetic Analysis of Two Evolutionarily Conserved miRNAs (miR166 and miR171) from Black Pepper (Piper nigrum L.)

Among computationally predicted and experimentally validated plant miRNAs, several are conserved across species boundaries in the plant kingdom. In this study, a combined experimental–in silico approach was adopted for characterization of two conserved miRNAs, miR166 and miR171, from black pepper (Piper nigrum). A PCR-based detection and cloning strategy of miRNAs from tissues of black pepper was used. Conservation analysis of miR166 and miR171 along with their corresponding targets identified from P. nigrum revealed that these miRNAs are highly conserved with their counterparts in other plant species. miRNA-mediated cleavage of the conserved targets was also verified by RLM-RACE experiments. Real-time quantitative PCR revealed the differential expression patterns of these miRNAs in black pepper tissues. Our miRNA-based phylogenetic analysis of plants belonging to the Piperaceae family was in agreement with the typical paleoherb evolutionary scheme of primitive angiosperms. This method will help in the detection of evolutionarily conserved miRNAs in other plant species and provide a strategy for a novel phylogenetic reconstruction based on the evolutionary history of miRNA genes.