Identification of Zinc Deficiency-Responsive MicroRNAs in Brassica juncea Roots by Small RNA Sequencing

The importance of zinc （Zn） as a micronutrient essential for plant growth and development is becoming increasingly apparent. Much of the world’s soil is Zn-deficient, and soil-based Zn deficiency is often accompanied by Zn deficiency in human populations. MicroRNAs （miRNAs） play important roles in the regulation of plant gene expression at the level of translation. Many miRNAs involved in the modulation of heavy metal toxicity responses in plants have been identiifed;however, the role of miRNAs in the plant Zn deifciency response is almost completely unknown. Using high-throughput Solexa sequencing, we identiifed several miRNAs that respond to Zn deifciency in Brassica juncea roots. At least 21 conserved candidate miRNA families, and 101 individual members within those families, were identiifed in both the control and the Zn-deifcient B. juncea roots. Among this, 15 miRNAs from 9 miRNA families were differentially expressed in the control and Zn-deifcient plants. Of the 15 differentially expressed miRNAs, 13 were up-regulated in the Zn-deifcient B. juncea roots, and only two, miR399b and miR845a, were down-regulated. Bioinformatics analysis indicated that these miRNAs were involved in modulating phytohormone response, plant growth and development, and abiotic stress responses in B. juncea roots. These data help to lay the foundation for further understanding of miRNA function in the regulation of the plant Zn deifciency response and its impact on plant growth and development.     

山羊卵巢富集chi-miR-100f-5p的鉴定与表达分析

【目的】探讨山羊卵巢中富集的miR-100及预测的靶基因与山羊卵巢功能及卵泡发育的潜在关系,为山羊繁殖机理研究提供数据。【方法】 以山羊卵巢组织为材料,通过miRNA的分离、克隆及测序得到山羊卵巢中表达的miR-100,采用RT-qPCR进行表达检测,用生物信息学方法预测miR-100的靶基因及其功能。【结果】① 山羊卵巢的测序结果中,一段成熟体为21nt 的序列与果蝇dan-miR-100高度同源,将其命名为chi-miR-100f-5p,其前体位于基因组负（-）链上,长度为59 bp,定位于山羊CM000899.1:33 997 894—33 997 914序列;② chi-miR-100f-5p 在山羊卵巢、垂体及心脏等组织中均有表达,说明此miRNA不是卵巢组织所特有;心脏和垂体组织中均有较高表达,其次是卵巢,而在肝脏、脾脏、肺及肾脏中表达显著低于卵巢（P ＜0.01）;在单产羔内蒙古绒山羊卵巢中chi-miR-100f-5p的表达极显著高于高繁殖力大足黑山羊卵巢（P＜0.01）;③ chi-miR-100f-5p主要靶作用于整合蛋白β1亚基基因及垂体特异性转录因子1基因,可能参与整合蛋白信号通路,促进垂体内分泌细胞的生长与分化,调节下丘脑-垂体-性腺轴激素分泌。【结论】山羊卵巢中新发现的chi-miR-100f-5p 可能是与繁殖调控有关的重要候选miRNAs 之一。     

用生物信息学挖掘玉米中的microRNAs及其靶基因

microRNA (miRNA)是一类内源性的、19~24碱基长度的小分子非编码RNA,通过碱基互补调控靶基因的表达,在多细胞生物的基因表达调控过程中扮演着十分重要的角色。植物中的miRNAs具有高度的保守性,这为通过同源比对发现保守的miRNAs提供了思路和途径。本研究通过对拟南芥、水稻等植物已知的miRNAs与玉米EST和GSS数据库的比对,并设置一系列严格的筛选标准,共筛选到23条新的玉米miRNAs;利用WMD 3在线植物miRNAs靶基因预测软件,对新发现的玉米miRNAs进行靶基因预测,总共预测到89个靶基因,进一步功能分析发现这些靶基因参与玉米的生长发育、信号转导、转录调节、新陈代谢及逆境胁迫响应等调控过程。     

苋菜试管开花过程中相关microRNA表达的qPCR分析

以苋菜无菌试管苗及其试管开花过程中各阶段培养物为材料,利用qPCR技术进行苋菜试管开花过程相关microRNA的表达分析。研究结果表明:由NormFinder软件计算出最适合的内参基因为ama-miR159a;在苋菜试管开花过程中,ama-miR156c、ama-miR156d、ama-miR156g相对表达量在幼苗期向壮苗期转变过程中呈下降趋势,并在整个发育过程中一直保持较低水平;ama-miR156h、ama-miR157b、ama-miR157c、ama-miR157d、ama-miR3和ama-miR10相对表达量在幼苗期向花芽期转变过程中呈下降趋势,并在花芽期达到最低;ama-miR172d的表达模式与miR156/miR157表达模式相反;苋菜试管苗幼苗期到花芽期形成时ama-miR319b相对表达量呈上升趋势,ama-miR164b在花芽期时相对表达量比较高,花芽期向开花期转变时,其相对表达量降低;ama-miR166a/miR166g表达在苋菜开花过程中始终呈上调表达;ama-miR167b/miR167d和ama-miR4相对表达量先略有升高然后降低,在花芽期降到最低,随后又升高。由此可见,microRNA在调控苋菜试管苗由幼苗期向壮苗期转变、营养生长向生殖生长转变以及花器官的形成等过程中起重要的调控作用。     

microRNAs在动物皮肤及毛囊发育中的调控作用研究进展

基因表达调控是动物被毛生长和发育的决定性因素。microRNA作为一种新发现的基因调控元件,在多种哺乳动物皮肤及毛囊中均有表达,并在转录后水平调节皮肤和毛发的生长和发育过程。从microRNA水平上解析绵羊、山羊及羊驼等被毛生长特点及发生机理,为提高毛用型经济动物的毛发品质和产量提供新的思路,同时也为更深入地研究皮肤组织中microRNA的功能提供更多的理论依据。作者主要针对目前已报道的在绵羊、山羊及羊驼等哺乳动物皮肤及毛囊发育中microRNA的调控作用进行综述。     

绿壳蛋鸡与白来航蛋鸡microRNA变异及其靶基因预测分析

本研究旨在分析绿壳蛋鸡与白来航蛋鸡microRNA变异及其靶基因,将已鉴定的microRNA与预测microRNA组成全基因组的microRNA,并通过与GGRS得到的两种鸡群体SNP位点进行映射,挖掘含SNP的microRNA。利用生物信息学的方法,对含SNP的microRNA进行靶基因预测,并对直接含有SNP的mature-microRNA进行聚焦。将这些靶基因进行富集,一共得到22个GO分类和10条KEGG信号通路与3个主要IPA调控网络,发现其在与生长相关的mTOR信号通路、Wnt信号通路、生长激素受体网络、胰岛素样生长因子Ⅰ受体网络得到了富集,与产蛋性状相关的卵母细胞减数分裂信号通路、黄体酮卵母细胞成熟信号通路得到了富集。此研究方法和结果可以给后期研究提供参考。     

microRNA与病毒关系研究进展

microRNA是一类广泛存在于真核生物细胞内的含20~22个核苷酸的非编码小RNA,它通过与目的mRNA 3'端非编码区的完全或不完全互补使得靶基因降解或翻译受阻。microRNA参与了众多基因的表达,涉及细胞的分化、发育、凋亡,病毒感染,免疫,机体代谢,肿瘤的发生等众多生命过程。microRNA和病毒的关系错综复杂,宿主细胞编码的microRNA可以对自身或病毒基因产生调控从而影响病毒感染,病毒编码的microRNA也可以对自身或宿主基因产生调控从而影响病毒感染,病毒也可以反过来影响宿主microRNA产生和作用。理清这两者之间的关系有助于科研人员发掘新的思路,更好地开展相关科研工作,为科学研究提供理论上的支持。因此,作者综述了microRNA的发现和命名、生成、作用机制,以及当前对于microRNA和病毒相互关系的新观点。     

利用Bac-to-Bac杆状病毒系统超量表达家蚕let-7簇microRNAs

【目的】利用Bac-to-Bac杆状病毒表达系统超量表达家蚕（Bombyx mori）let-7 簇（cluster）microRNAs：bmo-let-7、bmo-miR-100和bmo-miR-2795,为研究家蚕microRNA的功能提供参考。【方法】克隆家蚕let-7 miRNA簇（bmo-let-7 cluster,bmo-let-7-C）上各miRNA前体(pri-let-7、pri-miR-100与pri-miR-2795)和bmo-let-7-C全长序列,以红色荧光蛋白(red fluorescent protein,RFP)基因为报告基因,昆虫细胞表达载体pFastBac1为穿梭载体,通过Tn7转座子把目的基因和报告基因转座到杆状病毒A. californica nucleopolyhedrovirus (AcNPV)基因组上,获得各重组杆状病毒质粒（recombinant baculovirus plasmid,rBacmid）：Bac-let-7、Bac-miR-100、Bac-miR-2795和Bac-let-7-C。将重组Bacmid转染草地贪夜蛾（Spodoptera frugiperda）卵巢细胞系Sf9,72 h后用荧光显微镜检查红色荧光蛋白信号,荧光定量PCR检测miRNAs的表达。对转染的Sf9细胞进行、离心、收集上清液,获得具感染力的重组病毒,用于侵染新培养的Sf9细胞和注射家蚕幼虫个体,72 h后用荧光显微镜检查红色荧光蛋白信号,荧光定量PCR检测miRNAs的表达。【结果】成功将bmo-let-7-C上各miRNA前体和bmo-let-7-C全长序列构建到杆状病毒基因组上,获得了各miRNA及bmo-let-7-C的过量表达载体。将各重组过量表达载体分别转染草地贪夜蛾细胞系Sf9,72 h后在显微镜下观察到了红色荧光蛋白信号,荧光定量PCR检测结果表明各miRNA显著过量表达;通过离心收集的各miRNA重组过量表达病毒粒子感染新培养的Sf9细胞72 h后检测到了更强的红色荧光蛋白信号,定量PCR结果表明各miRNA均显著过量表达。将各miRNA的重组病毒注射到家蚕5龄1 d幼虫体内后,均能显著超量表达相应miRNA,但注射Bac-let-7-C后只有miR-2795显著过量表达,并有明显的组织差异性,在丝腺中没有过量表达,在脂肪体、血液和中肠中显著过量表达。【结论】利用杆状病毒过量表达系统在草地贪夜蛾细胞系Sf9和家蚕个体中超量表达了家蚕let-7簇miRNAs,为研究家蚕let-7簇和其他miRNAs的产生机制和功能提供了参考。     

Cold stress responsive microRNAs and their targets in Musa balbisiana

Cold stress is an environmental factor affecting plant development and production. Recently, microRNAs (miRNAs) have been found to be involved in several plant processes such as growth regulation and stress responses. Although miRNAs and their targets have been identified in several banana species, their participation during cold accumulation in banana remains unknown. In this study, two small RNA libraries were generated from micropropagated plantlets of Musa balbisiana grown at normal and low temperature (5°C). A total of 69 known miRNAs and 32 putative novel miRNAs were detected in the libraries by Solexa sequencing. Sixty-four cold-inducible miRNAs were identified through differentially expressed miRNAs analysis. Among 43 miRNAs belonging to 26 conserved miRNA families with altered expression, 18 were upregulated and 25 downregulated under cold stress. Of 21 putative novel miRNAs with altered expression, four were downregulated and 17 upregulated. Furthermore, eight miRNAs were validated by stem-loop qRT-PCR and their dynamic differential expression was analyzed. In addition, 393 target genes of 58 identified cold-responsive miRNAs were predicted and categorized by function. These results provide important information for further characterization and functional analysis of cold-responsive miRNAs in banana.     

花生根部耐盐相关miRNAs的鉴定与功能分析

miRNAs作为一种基因表达调控子在植物应答胁迫的过程中起着重要的作用,当花生受到盐胁迫时,也会有相应的miRNAs参与基因表达调控应答胁迫。本研究对200份花生品种进行萌发期耐盐性鉴定,获得了高耐盐花生品种3份,中耐盐花生品种5份,盐高敏感品种4份。并对不同耐盐性花生品种在盐胁迫处理条件下进行了抗氧化酶活性(SOD,POD,CAT)和MDA含量的测定。结果表明,耐盐性花生品种清除活性氧的能力大于盐敏感型。盐胁迫条件下,耐盐性花生植株MDA含量较少,受到的伤害相对较小,而盐敏感植株的受伤害程度最大,也证明了耐盐性花生品种在受到盐胁迫伤害时植物体内存在更强大的保护作用。通过小RNA测序及对靶基因序列进行功能分析和同源序列功能检索,获得了8条花生耐盐相关保守miRNAs序列,miR159-1,miR159-2,miR159-3,miR164-2,miR167-3,miR319-1,miR319-2,miR2111-1。荧光定量PCR测定结果表明,这些花生保守miRNAs受盐胁迫诱导,并调控其靶基因的应答反应。