MicroRNA参与植物花发育调控的研究进展

摘 要：MicroRNA(miRNA)是一类20到24nt的非编码小RNA,通过与靶mRNA序列的互补配对产生特异性,抑制了mRNA的表达或使其降解,从而调控靶基因的表达。本文主要综述了三类调控开花时间的miRNA家族成员：miR172,miR159 / miR319和miR156。其中,miR156主要调控植物生长周期转变;miR172通过调控AP2类基因,控制开花时间和花器官的形成;miR159和miR319的过量表达均会引起一些花发育障碍,如花期延迟。此外,还介绍了其它一些与花发育相关的miRNA,并对miRNA在花形成和发育中的研究方向进行了展望。     

Genome‐Wide Identification of MicroRNAs Responsive to High Temperature in Rice (Oryza sativa) by High‐Throughput Deep Sequencing

MicroRNAs (miRNAs) are known to play important roles in plant growth and stress response. Heat stress is a severe abiotic stresses by adversely affecting plant growth and yield. To identify heat‐responsive miRNAs at the genome‐wide level in rice (Oryza sativa), we constructed two small RNA libraries from young panicles treated or not with heat conditions. Ion torrent sequencing of the two libraries identified 294 known miRNAs and 539 novel miRNAs. Differential expression analysis showed that 26 miRNAs were downregulated and 21 miRNAs were upregulated in response to heat stress. Among them, five heat‐responsive miRNAs, including miR162b, miR529a‐p5, PC‐5P‐62245‐9, miR171b and miR169n, were validated by quantitative real‐time polymerase chain reaction. A total of 44 target genes of the differentially expressed miRNAs were predicted. These target genes are most significantly overrepresented in the cell growth process. The results demonstrated that rice miRNAs play critical roles in the heat stress response. This study opens up a new avenue for understanding the regulatory mechanisms of miRNAs involvement in the heat stress response in rice.     

毛白杨12种microRNAs的低温胁迫差异表达分析

为了探讨microRNAs (miRNAs)在杨树低温胁迫不同时间段的差异表达规律,初步鉴定参与杨树低温胁迫响应的miRNAs,以重要乡土树种毛白杨为材料,进行不同时间段(0、8、14、20 h)的低温胁迫(4℃)处理,分别提取小片段RNAs (<200 nt),利用实时定量PCR技术研究毛白杨在低温胁迫后12种miRNAs的差异表达规律。结果显示,miRNAs的表达在低温胁迫下有显著变化,且呈现动态反应。在低温胁迫下,大部分miRNAs的表达受到抑制。miR168a、miR169ac、miR394a-3p、miR530a的表达量在各个时间段显著下调。该研究初步表明,miRNAs在调控毛白杨对低温胁迫的反应中起着重要作用。     

Identification and validation of cold responsive microRNAs of <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> using high throughput sequencing

Cold stress is one of the major abiotic factors that influence the productivity and geographical distribution of many agriculturally important crops like Hevea brasiliensis. Cultivation of H. brasiliensis in India is being extended to northeastern regions, where low temperature during winter adversely affects its survival, growth, and productivity. Developing cold-tolerant genotypes is a primary requisite to maximize the productivity under such challenging environmental conditions. However, lack of methods for early evaluation of cold tolerance in the newly developed clones and the extensive time required for assessing their tolerance in the field are major constraints for clonal selection. The present study was initiated with an objective to identify and characterize cold stress responsive miRNAs from H. brasiliensis that show stronger association with cold tolerance. Next generation sequencing using Illumina HiSeq method revealed the expression of 21 and 29 conserved miRNA (from clone RRIM 600) families in cold-stressed and control samples, respectively. Forty-two novel miRNAs were identified from this study. Upon differential expression analysis, eight conserved miRNAs were found commonly expressed in both the samples. When expression analyses were performed subsequently with six selected miRNAs in two Hevea clones (viz. RRII 105 and RRIM 600), miR169 showed a strong association with cold tolerance. miRNAs such as miR482 and miR159 also exhibited association with cold tolerance. This study suggests the possibility of employing these miRNAs as markers for cold tolerance after validation in more number of genotypes with varying levels of cold tolerance.     

Old and New Cultivars of Soya Bean (Glycine max L.) Subjected to Soil Drying Differ in Abscisic Acid Accumulation,Water Relations Characteristics and Yield

Two old (Huangsedadou and Longxixiaohuangpi (LX)) and two new (Jindou 19 (JD) and Zhonghuang 30 (ZH)) soya bean (Glycine max (L.) Merr.) cultivars were used to investigate the influence of soil drying on the abscisic acid (ABA) accumulation in leaves, stomatal conductance (g_s), leaf water relations, osmotic adjustment (OA), leaf desiccation tolerance, yield and yield components. The greater ABA accumulation was induced by soil drying, which also inducing g_s decreased at higher soil water contents (SWC) and leaf relative water content (RWC) significantly decreased at lower SWC in the new soya bean cultivars than in the old soya bean cultivars. The soil water threshold between the value at which stomata began to close and the RWC began to decrease was significantly broader in the new cultivars than in the old cultivars. The new cultivars had significantly higher OA and lower lethal leaf water potential than old cultivars when the soil dried. The old cultivars had greater biomass, but lower grain yield than the new cultivars in well‐watered, moderate stress and severe stress conditions. Thus with soil drying, the new soya bean cultivars demonstrated greater adaptation to drought by inducing greater ABA accumulation, stomatal closure at higher SWC, enhanced OA and better water relations, associated with increased leaf desiccation tolerance, greater water use efficiency and higher yield.     

植物miR398家族的分子特征与进化分析

miR398家族是植物界中一类保守的miRNA家族,本文旨在探究植物miR398家族成员的分子特征,阐释其系统进化规律。以数据库公布的植物miR398成熟体、前体及靶标序列为供试数据,用统计学和生物信息学法分析其分子特征与进化规律。miR398家族成员在34个植物物种中分布广泛,其成熟体长度、核苷酸种类、前体茎环结构及对靶基因抑制类型均符合典型的植物miRNA分子特征。不同植物间miR398成熟体保守性高于前体,前体茎部保守性高于环部。同一物种间的miR398家族成员具有不同的进化速率,少数物种间的miR398家族具有协同进化特征。靶基因功能预测结果显示,miR398家族成员多以切割抑制的方式参与植物的逆境胁迫响应。植物miR398家族兼具保守性与多态性特征,不同植物间的miR398家族成员具有多样的进化模式。研究结果为miR398介导的植物逆境胁迫响应机制探究奠定理论基础。     

香蕉冷胁迫相关microRNA差异表达分析

研究香蕉miRNA在冷胁迫处理下的动态表达,初步探索miRNA在香蕉冷胁迫过程中的可能作用。利用茎环引物实时荧光RT-PCR (stem-loop qRT-PCR)策略,检测冷胁迫响应miRNA在5℃冷胁迫处理不同时间点香蕉幼苗叶片中的表达模式。结果表明,在检测的15个香蕉miRNAs中,不同的miRNA 在同一冷胁迫时间下的表达量存在很大差异。根据其表达模式,可将15个miRNA分成4组。组1 和组2中的12个miRNA (miR159e, miR159f, miR160-3p, miR397a, miR399j, miR164e, miR444a, miR408, miR5179, miR530, miR535和miR5538),在5&;#176;C冷胁迫处理下表达量总体为上调,其在胁迫过程中的平均表达水平高于对照CK (0 h) ,说明冷胁迫诱导此类miRNA的表达。第3组包括miR156l、miR162a和miR166a,其在5℃冷胁迫处理各时间点的表达量均低于对照,为下调表达,说明冷胁迫抑制此类miRNA的表达。第4组仅含一个miRNA,即miR156a,除在冷胁迫处理12 h表达量明显上调外,其余时间点均下调表达。对这些miRNAs 作用的靶基因进行预测和分析发现,它们主要参与植物的新陈代谢、信号传导和生长发育过程等。这为进一步验证与低温胁迫相关的目的miRNA 的功能提供理论依据。     

利用solexa测序技术发掘木薯microRNAs

为了发掘木薯中的microRNAs (miRNAs),了解其在木薯中的调控机制,本研究利用Solexa测序技术在木薯中发现了大量的miRNAs,并利用一种新的miRNA定量检测技术（Multiplexed RT法）对其进行实验验证。通过对构建的3个小RNA文库进行solexa测序与生物信息学分析,最终获得19个家族93个保守的miRNA和74个新的miRNA,实验验证了测序获得的83个miRNA。在Arg7块根中特异表达的有6个miRNA,分别为miR172d、miR396c、miR398a、miR398b、miR399e、miR399f,叶片中特异表达的有13个,须根中特异表达的有3个,这些miRNAs将为深入了解木薯块根发育和淀粉累积机理提供了一个新的视角。     

Responses of Soya bean Yield and Yield Component Distribution across the Main Axis under Source–Sink Manipulation

Extensive studies have been conducted regarding the source–sink alterations on soya bean [Glycine max (L.) Merr.] yield, but limited information is available for the seed yield and distribution of yield components across main nodes under whole‐plant light‐enriched conditions. A 2‐year research was conducted at the University of Massachusetts Agronomy Farm under ambient and light‐enriched conditions for two old and two new cultivars planted with a normal density. A randomized block design was used in each year. Light enrichment was initiated at the onset of flowering by installing a 90‐cm tall wire mesh fencing (mesh hole size 4–5 cm) adjacent to the centre row and sloping away at a 45° angle and was left in place for the remainder of the growing season. Five source–sink manipulations were initiated following the establishment of light enrichment. The yield sensitivity of the two old cultivars to the changes in source strength and light‐enriched conditions during reproductive period was much greater than that of the two new cultivars. The increased yield by light enrichment in various manipulations of source–sink treatments was, in part, due to increased branch contribution. Most pods were produced at the nodes in the middle sections of the plants and appeared in higher node position in new cultivars compared with the old cultivars. Seed number per pod and seed size was quite uniform across all node positions. Seed size of both the old and the new cultivars was responsive to changes in the source–sink ratio and changes in the environment during the growth of the soya bean plant. The results suggested that soya bean plants showed several mechanisms to control or realize their excess reproductive potential in a constantly changing environment.     

Yield–Density Relation of Glyphosate-Resistant Soya beans and their Responses to Light Enrichment in North-eastern USA

Optimum plant population densities are a key means of achieving higher seed yield in soya bean [Glycine max (L.) Merr.]. Limited information is available on yield‐density relation of glyphosate‐resistant soya beans in north‐eastern USA. The objective of this research was to determine the appropriate populations for glyphosate‐resistant soya beans, and if the yield potential of glyphosate‐resistant soya bean produced in light‐enriched conditions was affected by populations. Eight glyphosate‐resistant soya bean cultivars with three populations (300 000, 500 000 and 800 000 plants ha⁻¹) were grown under both ambient and light‐enriched conditions in 2002 and 2003. Yield of all cultivars responded to density linearly. As density increased, grain yield was increased by up to 92 % among cultivars. Light enrichment increased yield for all cultivars across the 2 years, although some cultivars were more sensitive. Harvest index either remained unchanged or declined slightly at higher density in 2002, and there was no difference among treatments in 2003. Both pod number and seed number per plant were significantly decreased with the increase of density across the 2 years, while seeds per pod declined slightly or remained unchanged. Greater seed size was obtained in higher density with varied degree depending on cultivars across the 2 years except for those cultivars with relatively larger seed. The increase in seed size by light enrichment was cultivar and density dependent, and varied between years. 800 000 plants ha⁻¹ could be a suitable practice in producing higher yield in north‐east USA for glyphosate‐resistant soya bean. Maintaining the mass of an individual seed is an important strategy in achieving high yield at high population. Establishing mechanisms responsible for the greatest yields via high population under light‐enriched conditions, may provide insights for management and phenotypic improvement.     

Soya Bean (Glycine max L. Merr.) Genotype Response to Early-season Flooding: II. Aboveground Growth and Biomass

Soya bean is often grown in regions prone to periodic flooding, thus selecting cultivars that maintain production under waterlogged conditions is desirable. An experiment involving flooded soya beans was planted in southern Florida to examine (1) stem and leaf growth; (2) morphological adaptations; and (3) the relationship between early‐season and late‐season flood tolerance in flooded soya beans. Eleven soya bean genotypes previously defined as tolerant or sensitive to flooding were subjected to three treatments at 21 days after sowing (DAS): (1) no flood, (2) 2‐week flood and (3) 4‐week flood. All plants were harvested 49 DAS. Flooded plants exhibited lower stem dry weights but greater partitioning to the stem. Non‐flood treatments had greater leaf dry weight, leaf area and partitioning to leaves than flooded plants. There were positive correlations of genotype stem dry weight and leaf dry weight to early‐season flood tolerance but stem partitioning was negatively correlated with early‐season flood tolerance. Genotypic rankings of early‐season flood tolerance in this study were not correlated with earlier studies basing flood tolerance on seed yield. Our study highlights the range of soya bean morphological adaptations in response to flood. However, our results indicate that early‐season screening may not be an accurate predictor of soya bean genotypic response to late‐season flood.     

Aerial canopy temperature differences between fast‐ and slow‐wilting soya bean genotypes

Drought stress limits crop growth and yield in soya bean (Glycine max [L.] Merr.), but there are relatively few tools available to assess the ability of different genotypes to tolerate drought. Aerial infrared image analysis was evaluated as a potential tool for identifying drought tolerance in soya bean. Drought effects were evaluated from late vegetative to mid‐reproductive stages of soya bean development in an experiment with ten genotypes including five slow‐ and five fast‐wilting genotypes that were from a population derived from Benning×PI416937. There were two deficit irrigation levels for 2 years and one deficit irrigation level for the third year along with a fully irrigated control level. When the canopy was completely closed, relative canopy temperature was determined using an infrared camera taken from an aerial platform 50–75 m above the experiment. As water availability decreased, the relative canopy temperature generally increased. Moreover, slow‐wilting soya bean genotypes generally had lower canopy temperature compared to fast‐wilting genotypes, and grain yield was generally positively associated with cool canopy temperatures. The results indicate that the determination of canopy temperature is a promising tool for rapid characterization of drought‐related traits in soya bean.     

半定量RT-PCR方法研究microRNA393在旱处理后的孕穗期水稻叶片和穗部的表达情况

microRNAs是新近发现的一类具有重要功能的小分子RNA.研究表明,一些miRNAs与植物抗逆反应相关.本文使用半定量RT-PCR的方法,研究了miR393在旱处理后的孕穗期水稻叶片和穗部的表达情况.结果发现,miR393在胁迫后的叶片和穗部表达上调.同时也发现,miR393在胁迫前后穗部的表达略高于其在叶片中的表达.实验结果也表明,半定量RT-PCR适用于分析miRNAs的表达.     

陆地棉种子发育过程中 microRNA 的挖掘与功能研 究简

 microRNAs (miRNAs)广泛参与调控植物的生长和发育,但这类分子是否对棉花胚珠和纤维的生长发育起到重要的作用,还有待验证。对这些小分子的认识,一般先要从序列信息的获得上开始。应用基因芯片对TM-1 +5 DPA （开花后5 d）胚珠总RNA进行了miRNA 的筛选研究。结果显示,含有352 个探针的芯片成功钓取到199 个陆地棉miRNA,其中绝大多数为首次报道。此外,对miR399 研究发现,它与纤维中磷的含量变化相关; miR169 可能与干旱应激反应有关。     

小金海棠缺铁胁迫相关miR394a的克隆与表达分析

为了研究铁高效植物小金海棠的miRNAs信息及在缺铁处理下miRNA的表达变化,以进一步了解小金海棠缺铁分子调控机制。以改良CTAB法提取的总RNA为模板,从小金海棠中分离得到miR394a。通过Real-time PCR检测miR394a的表达,并利用生物信息学方法预测了miR394a的靶基因及其功能。miR394a具有高度保守性,从小金海棠中分离的miR394a与其他物种的miR394a序列高度相似。缺铁胁迫后,小金海棠的miR394a在根及叶中都有表达,但表达模式有所不同,miR394a在根部响应较为迅速,缺铁处理1天时,即有上调表达;而在叶片的响应较晚,缺铁3天时有上调表达。miR394a的靶基因预测表明,miR394a主要调控转录因子及参与代谢途径的基因。结果表明,miR394a受缺铁胁迫所诱导,参与了小金海棠缺铁调控途径,在缺铁调控中起重要作用。     

苹果炭疽病抗性miRNA的筛选

为筛选炭疽病菌侵染苹果属植物后相关miRNA的响应情况,以抗病品种‘八棱脆’海棠、感病品种‘嘎啦’苹果2个为试验材料,分别用胶胞炭疽孢子悬浮液侵染组培苗,4天后发病,提取植物总RNA并反转miRNA,用qRT-PCR分析2种植物中抗病相关miRNA相对表达量的差异,并对其靶基因进行预测。在抗性品种中miR390a和miR396b/c/f表达量下调,而在感病品种中表达量上调,且其靶基因均有LRR受体样丝氨酸/苏氨酸蛋白,miR482b靶基因为抗性蛋白。miR390a、miR482b和miR396b/c/f可能在苹果炭疽病的侵染过程中起重要作用。     

大豆黑河5号和Fiskeby 5号种子对干燥脱水敏感性的差异及分析

在同样干燥条件下，中英两个大豆品种对脱水敏感性差异很大：中国品种黑河5号超干后的干裂率高达87％，而英国品种Fiskeby5号则只有17．6％的破裂率。去种皮实验证明：种皮虽对防止大豆种子干燥破裂和吸胀损伤甚为重要，但决定大豆品种间干燥破损度是异的主要因素是品种固有的遗传特性。     

Effects of NaCl on Root Growth and Cell Wall Composition of Two Soya bean Cultivars with Contrasting Salt Tolerance

Our objectives were to determine the influence of salinity on root cell wall composition in soya beans and the possible mechanism of salt tolerance. Two soya bean cultivars, Touzan 69 (salt sensitive) and Dare (salt tolerant), were selected as experimental material for comparison. Root growth was clearly inhibited by salinity in both cultivars, but Touzan 69 showed more severe reductions in root length than Dare. In the 0–5 mm root segment (from root tip), the total cell wall sugar content of Touzan 69 decreased considerably due to salinity as were the pectin, hemicellulose and cellulose fractions. In Dare, NaCl treatments only caused a slight decrease in the pectin fraction and no marked change in hemicellulose and cellulose fractions. Without salt treatment, the pectin fraction accounted for about 40 % and cellulose for 30 % of cell wall composition in the 0–5 mm root segment; in the 5–10 segment (from root tip), pectin and cellulose accounted for 27 % and 45 % in Touzan 69, and 34 % and 38 % in Dare. The percentage of pectin decreased and that of cellulose increased in the 5–10 mm root segment compared with the 0–5 mm segment. This indicates that pectin largely regulates cell growth, as the 0–5 mm region is considered the elongation zone of soya bean roots. Salt treatment decreased the percentage of pectin, but increased that of cellulose across root zones of the two cultivars, suggesting that salt presence may increase cell wall rigidity, and thus, inhibits root growth. Dare was able to maintain its main root cell wall substances, an apparent advantage for root cell growth that may overall improve its salt tolerance. Also, the less reduction in cell wall uronic acid was of some benefit in the positive regulation of root cell growth in Dare. The changes in cell wall composition, especially the pectin content had a close relation with the regulation of root growth. The difference in salt tolerance between the two tested cultivars can partly be explained on the basis of these changes in response to salinity. Sugar compounds in each cell wall constituent and their functions in ion transport as well as the relationship between root cell wall and soya bean salt tolerance need to be further investigated.     

外源褪黑素对低温胁迫下菜豆种子萌发及抗性的影响

为了探究褪黑素(Mt)对低温环境下菜豆种子的影响,本研究对菜豆种子进行低温处理(LT)和低温加褪黑素的处理(LT+Mt),并设置常温处理作为对照(CK),对同时期的各项生理指标进行测定。结果表明：Mt可以提高低温胁迫下的种子萌发率,但萌发率要低于对照;同时对渗透调节物质、O₂^-和MDA、抗氧化酶活性进行测定,发现低温增加了渗透调节物质的含量,当加入Mt后其含量进一步增加;低温处理后O₂^-和MDA的含量增加,当加入Mt后其含量明显降低,但是要高于对照;低温处理后抗氧化酶活性略低于对照,当加入Mt后其含量显著升高。研究表明外源Mt有效的减轻了低温对种子萌发的损害,从而促进种子萌发过程中抵御低温的能力。