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。     

半夏miR167及其靶基因响应大豆花叶病毒胁迫的表达

MicroRNAs(miRNAs)是一类小的非编码RNA,在植物逆境及生物胁迫中通过调节靶基因来发挥重要的调控作用。半夏内源miR167是否参与了大豆花叶病毒(soybean mosaic virus,SMV)的胁迫及相互作用关系尚不清楚。本研究利用茎环法扩增半夏内源miR167基因，命名为pt-miR167,采用生物信息学软件对其进行保守性分析，系统进化性分析及靶基因预测。在此基础上，采用qRT-PCR技术检测病毒积累量、miR167及其潜在的靶基因的表达水平，分析miR167及靶基因响应病毒侵染的表达模式。结果表明，病毒积累量在5～15 d时迅速增加，其中10 d时增加最快，随后呈现缓慢上升趋势。病毒侵染后，pt-miR167相比对照组，表达量呈下调，并且在10 d时表达量最低。进化树分析表明，pt-miR167与番茄(Solanum lycopersicum)中的sly-miR167a聚为一族，高度同源。预测的潜在主要靶基因ARF6表达量与pt-miR167表达量呈现正好相反的趋势，其在10 d时表达量达到最高。本研究表明，miRNA167参与了寄主半夏与病毒SMV的相互作用关系...     

普通野生稻miR160f的克隆和功能分析

MicroRNAs是一类在调节基因转录后表达中起重要作用的非编码RNA。miR160通过调节生长素响应因子(ARF)参与根细胞的分裂和分化,从而影响根的发育。克隆了普通野生稻mi R160f基因,并将其转入拟南芥中鉴定功能。结果表明,过表达mi R160f的拟南芥莲座叶的数量减少,抽薹时间缩短,开花的时间提前。qPCR检测显示miR160f的靶基因ARF10、ARF16及ARF17在过表达拟南芥植株中的表达下调,而ARF10和ARF16蛋白的缺失或减少会导致根冠细胞分化受阻、分裂失控,并导致根尖干细胞群的异位扩大,因此可以表明miR160不仅影响根的发育,还可能与水稻的开花时间相关。     

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。     

植物miR390的研究进展

MicroRNAs(miRNAs)是真核生物中一类非编码内源小分子RNA,它通过对靶m RNA的剪切或抑制靶m RNA的翻译来调控基因的表达,从而对靶基因实施转录后水平调控,在植物器官形成、生长发育、信号转导及非生物胁迫应答等过程起重要作用。MicroRNA390(miR390)家族是一个古老的高度保守的家族,其主要的靶基因AGO7是RNA沉默复合体的重要组成成分,广泛参与对靶miRNA的剪切,可能在植物的生长发育、侧生器官极性形成、花器官形成及胁迫等方面有重要作用,但是目前对miR390的研究主要集中在植物生长发育方面,在非生物逆境胁迫应答方面鲜有报道。综述了miR390的发现及其在植物中的类型、miR390家族的形成过程及miR390参与植物的生长发育过程和响应重金属、干旱、盐、低温等非生物胁迫的作用,同时对miRNAs功能研究手段作了展望,有利于进一步综合了解miR390的研究概况及对miR390参与非生物胁迫的研究。     

光皮桦miR164及其靶基因NAC1在低氮胁迫中的表达分析

氮是植物生长发育所必需的大量营养元素,植物缺氮后严重影响地上部分生物量的积累,因此,揭示植物如何抵抗或适应低氮胁迫的分子机制具有重要意义。杨树(Populus tremula × P. alba)NAC1(NAM, ATAF, CUC 1)基因位于调控网络上游,在低氮环境下调控下游关键基因的表达,进而调控根系生长以抵抗低氮胁迫。本文以光皮桦(Betula luminifera)G49-3无性系组培苗为材料,探讨了miR164及其靶基因NAC1对低氮胁迫的响应。通过RACE技术克隆了光皮桦NAC1基因(GenBank登录号：KT900889),全长1497 bp,编码358个氨基酸,N端具有高度保守的NAM结构域;运用5′-RACE验证了NAC1为miR164靶基因,切割位点在第10和11位碱基之间;采用qRT-PCR分析miR164与靶基因NAC1在低氮胁迫时的表达模式,发现miR164表达在根中的低氮处理前期(4 d)受到抑制,而后升高,而茎叶中表达模式与根不同;靶基因NAC1与miR164表达水平呈负相关,且在恢复实验组(重新添加全营养液)中,根中miR164表达上升,NAC1显示出相应的表达变化,暗示miR164及其靶基因NAC1可能在低氮胁迫响应中发挥调控功能。本研究结果有助于揭示miR164对NAC1在低氮胁迫响应中转录后水平的分子调控机制,为进一步研究miR164-NAC1在低氮胁迫响应中的功能提供有价值的信息。     

miR172-AP2模块调控植物生长发育及逆境响应的研究进展

MicroRNA (miRNA)是一类具有调控能力的非编码小分子RNA, 通过与靶基因mRNA特异或非特异性结合, 诱导靶基因mRNA降解或抑制其翻译, 从而调控植物的生长发育。其中, miR172的靶基因AP2所编码的转录因子为植物所特有, miR172在转录后或翻译水平对AP2进行表达调控, 进而调控植物的花发育、时序转换、小穗形态、块茎和果实发育、结瘤(豆科)以及逆境响应等过程。该文综述了近年来miR172-AP2模块在植物生长发育调控方面的最新研究进展。     

甜杨低温响应microRNAs的克隆与分析

MicroRNAs (miRNAs)作为一类21碱基左右的非编码小RNAs,参与植物生长发育的调控,并在植物对生物与非生物胁迫的应答过程中发挥重要作用.本研究依据miRNA高度保守特点,利用已公布的毛果杨(Populus trichocarpa)基因组序列设计引物,从甜杨(Populus suaveolens)基因组中克隆获得了12个miRNA基因座序列.序列比对结果表明,这些miRNA基因均为毛果杨低温响应miRNA基因的同源序列.同时,以低温(0℃)处理0～48 h的甜杨幼苗为试材,通过半定量RT-PCR法对miRNA基因的成熟体序列在不同处理时间下的表达谱进行分析,结果显示,大多数miRNA成熟体序列在甜杨低温胁迫下的表达模式与其在毛果杨中的表达极为相似,由此可推测这些保守性miRNAs可能在甜杨和毛果杨两物种对低温胁迫的应答反应中发挥相似的功能,而miR168a、miR168b和miR475a在两物种间表达现象的差异,表明它们可能通过调控多种靶基因而发挥不同作用.本文结果将为进一步研究甜杨基因功能提供基础.     

5个葡萄microRNAs及其靶基因在冬芽二次成花过程中的表达

试验以‘藤稔’葡萄为材料,利用荧光定量PCR技术检测5个葡萄miRNAs( vv-miR160a、vv-miR171a、VVmiR159、vv miR164c和vv-miR167c)及其靶基因在不同摘心处理的冬芽发育过程中的动态表达特征,以探讨microRNAs调控花发育的分子机理.结果显示:5条miRNAs及其靶基因在二次成花的冬芽中的表达发生了明显变化.其中,vv-miR160a、vv miR171a和vvmiR159表达水平明显增强,在花序中有最高表达;vv-miR164c和VVmiR167c的表达水平明显减弱,在花序中的表达最低或较低,而这些vv-miRNAs在对照组中表达水平均无明显变化.研究表明,上述miRNAs参与了葡萄冬芽的二次花的发育;从miRNAs与其靶基因的表达呈现的消长变化趋势看,这些vvmiRNAs通过负调控其靶基因表达而起作用.     

低温胁迫下嫁接对黄瓜叶片SOD和CAT基因表达与活性变化的影响

研究了低温胁迫下嫁接和自根黄瓜叶片Mn-SOD、Cu/Zn-SOD和CAT mRNA基因表达和酶活性变化及其与抗冷性的关系.结果表明:低温胁迫下,嫁接与自根黄瓜叶片Cu/Zn-SOD、Mn-SOD mRNA基因相对表达量变化分别与其Cu/Zn-SOD、Mn-SOD活性变化相吻合,而CATmRNA相对表达量变化与其CAT活性变化并不一致;嫁接黄瓜叶片Cu/Zn-SOD和Mn-SOD mRNA相对表达量及SOD、Cu/Zn-SOD和Mn-SOD活性均高于自根黄瓜,MDA含量和电解质渗漏率均低于自根黄瓜,嫁接黄瓜较高的SOD基因表达量调控的较高SOD活性是其抗冷性强于自根黄瓜的主要因素;嫁接黄瓜的功能叶CAT mRNA相对表达量略高于自根黄瓜,而幼叶CAT mRNA相对表达量低于后者,但两者CAT活性差异不大,说明低温胁迫对嫁接黄瓜叶片CAT mRNA相对表达量及CAT活性的影响不大.     

Osa‐miR167d facilitates infection of Magnaporthe oryzae in rice

MicroRNAs (miRNAs) play important roles in rice response to Magnaporthe oryzae, the causative agent of rice blast disease. Studying the roles of rice miRNAs is of great significance for the disease control. Osa‐miR167d belongs to a conserved miRNA family targeting auxin responsive factor (ARF) genes that act in developmental and stress‐induced responses. Here, we show that Osa‐miR167d plays a negative role in rice immunity against M. oryzae by suppressing its target gene. The expression of Osa‐miR167d was significantly suppressed in a resistant accession at and after 24 h post inoculation (hpi), however, its expression was significantly increased at 24 hpi in the susceptible accession upon M. oryzae infection. Transgenic rice lines over‐expressing Osa‐miR167d were highly susceptible to multiple blast fungal strains. By contrast, transgenic lines expressing a target mimicry to block Osa‐miR167d enhanced resistance to rice blast disease. In addition, knocking out the target gene ARF12 led to hyper‐susceptibility to multiple blast fungal strains. Taken together, our results indicate that Osa‐miR167d negatively regulate rice immunity to facilitate the infection of M. oryzae by downregulating ARF12. Thus, Osa‐miR167d‐ARF12 regulatory module could be valuable in improvement of blast‐disease resistance.     

AMF与嫁接对西瓜连作土壤理化和微生物状况的影响

将嫁接京欣1号葫芦砧Lagenaria siceraria、京欣2号南瓜砧Cucurbita maxima × Cucurbita moschata和不嫁接的京欣4号西瓜自根苗定植于西瓜连作土壤中,接种丛枝菌根真菌（arbuscular mycorrhizal fungi,AMF）摩西管柄囊霉Funneliformis mosseae、变形球囊霉Glomus versiforme和根内根孢囊霉Rhizophagus intraradices处理,以不接种丛枝菌根真菌的处理为对照。结果表明,与对照相比所有处理均显著降低土壤全氮、速效磷、速效钾和有机质含量,但提高土壤pH。接种AMF并嫁接处理的>2mm和2-0.25mm粒级土壤团聚体含量显著高于单纯嫁接或只接种AMF或对照,所有处理的0.25-0.053mm与<0.053mm粒级土壤团聚体含量均显著低于对照。以接种AMF并嫁接处理的土壤细菌数量显著高于其他处理,而所有处理的真菌数量则显著低于对照,接种AMF并嫁接处理的放线菌数量一般高于对照。接种AMF并嫁接处理的土壤蔗糖酶、多酚氧化酶和脲酶活性显著高于对照。接种AMF结合嫁接改善连作土壤理化特性和土壤微生物区系的效应大于只嫁接或仅接种AMF的单一处理的。     

低温胁迫对嫁接西瓜耐冷性和活性氧清除系统的影响

研究了西瓜实生苗和以黑籽南瓜、超丰F1为砧木的嫁接苗的耐冷性及活性氧清除系统的差异．结果表明,低温胁迫下,嫁接苗的耐冷性明显高于实生苗,表现为以黑籽南瓜为砧木的嫁接苗的耐冷性>以超丰F1为砧木的嫁接苗>实生苗,此外嫁接苗和实生苗均表现为叶片中叶绿素含量下降,丙二醛(MDA)含量上升,非酶促抗氧化剂抗坏血酸(AsA)、谷胱甘肽(GSH)含量和抗氧化酶超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(AsA-POD)、脱氢抗坏血酸还原酶(DR)活性下降,说明低温逆境降低了植物体防御活性氧有关的酶促和非酶促保护系统能力,提高了体内自由基浓度,加剧了膜脂过氧化．嫁接苗的活性氧清除能力均高于自根苗,且嫁接苗中耐冷性越强的活性氧清除能力越高,说明西瓜嫁接后耐冷性的提高是与植物体内活性氧清除系统中抗氧化剂含量和抗氧化酶活性提高有关。     

Prevalence of aberrant methylation of p14ARF over p16INK4a in some human primary tumors

The INK4a/ARF locus encodes two unrelated tumor suppressor proteins, p16INK4a and p14ARF, which participate in the two main cell-cycle control pathways, p16–Rb and p14–p53. Methylation of CpG promoter islands has been described as a mechanism of gene silencing. Exon 1 of the p16INK4a gene and the p14ARF promoter gene reside within CpG islands. Therefore, both can become methylated de novo and silenced. It has recently been proposed that the methylation changes in certain genes could be used as molecular markers for the detection of almost all forms of human cancer. Here, we analyzed concomitantly in each tumor sample and normal tissue the methylation status of p16INK4a and p14ARF by methylation-specific PCR (MSP) in 100 breast, 95 colon and 27 bladder carcinomas. A series of clinicopathological parameter were obtained from the medical records of the patients, p14ARF showed a higher rate of hypermethylation than p16INK4a in all three tumor types. p16INK4a and p14ARF aberrant methylation was significantly correlated with poor prognosis clinicopathological parameters of the three tumor types. We conclude that both p16INKa and p14ARF hypermethylation may be involved in breast, colon and bladder carcinogenesis, with special emphasis on the role of the lesser studied p14ARF gene, and that tumors with aberrant methylation in the two genes were associated with worse prognosis.     

A set of Arabidopsis thaliana miRNAs involve shoot regeneration in vitro

Plant miRNAs, the critical regulator of gene expression, involve many development processes in vivo. However, the roles of miRNAs in plant cell proliferation and redifferntiation in vitro remain unknown. To determine better the molecular mechanism of these processes, we have recently reported that a set of miRNAs with different expression patterns between cells of totipotent and non-totipotent Arabidopsis calli. Some of these were specifically up- or downregulated during callus formation or shoot regeneration, and other development. Among them, miR160, and one of its target genes, ARF10, regulated Arabidopsis in vitro shoot regeneration via WUS, CLV3 and CUC1/2. The miR160-overexpressing, 35S transgenic lines, exhibited reduced shoot regeneration efficiency. The mARF10, a miR160-resistant form of ARF10, showed a high level of shoot regeneration ability. In the transgenic, expression of the above shoot meristem-specific genes was elevated, which is consistent with the improved shoot regeneration. In contrast, the ARF10 deficient knockout mutant produced fewer regenerated shoot. However, overexpressors of ARF10 were only marginally more efficient than the wild type with the respect to shoot regeneration. Our observation strongly supports that proper shoot regeneration from in vitro cultured cells requires the miR160-directed negative influence of ARF10. The enhanced expression of ARF10 is likely to have contributed to the improved regeneration ability.     

Overexpression of AtTTP Affects ARF17 Expression and Leads to Male Sterility in Arabidopsis

Callose synthesis is critical for the formation of the pollen wall pattern. CalS5 is thought to be the major synthethase for the callose wall. In the Arabidopsis anther, ARF17 regulates the expression of CalS5 and is the target of miR160. Plants expressing miR160-resistant ARF17 (35S:5mARF17 lines) with increased ARF17 mRNA levels display male sterility. Here we report a zinc finger family gene, AtTTP, which is involved in miR160 maturation and callose synthesis in Arabidopsis. AtTTP is expressed in microsporocytes, tetrads and tapetal cells in the anther. Over-expression lines of AtTTP (AtTTP-OE line) exhibited reduced male fertility. CalS5 expression was tremendously reduced and the tetrad callose wall became much thinner in the AtTTP-OE line. Northern blotting hybridization and quantitative RT-PCR analysis revealed that miR160 was decreased, while the expression of ARF17 was increased in the AtTTP-OE line. Based on these results, we propose that AtTTP associates with miR160 in order to regulate the ARF17 expression needed for callose synthesis and pollen wall formation.