The choice of target site is crucial in artificial miRNA-mediated virus resistance in transgenic Nicotiana tabacum

MicroRNAs (miRNAs) are negative regulators of gene expression via mRNA degradation or translational repression. The potential of artificial miRNAs (amiRNAs) as antiviral agents has been used in plant biotechnology. In this study, we designed eight amiRNAs derived from Potato Virus Y (PVY) Coat Protein RNA sequences and generated transgenic tobacco plants to express these amiRNAs to confer virus resistance against PVY. Together with transient assays, the hairpin amiRcp precursors that mimic natural miRNA precursor molecules proved to be effective in expressing amiRcps and silencing the target gene. Virus resistance assay revealed that not all amiRcps targeting viral CP sequence are equally effective in preventing PVY infection. Plants with amiRcp-8 targeting the 3′ end (nt735–nt754) region exhibited high virus resistance up to 64.69%. The amiRcp-6 harboring RNA sequence (nt567–nt586) induced the lowest percentages with only 17.05%. Besides, northern blots showed that there was a correlation between the resistance level and the accumulation of amiRNA expression. Furthermore, we used bioinformatics approach to predict the mRNA structure and found that targeting sequences of loose structure benefit to improve the virus resistance level. Our results indicate that the selection of appropriate target sequence is crucial for transgenic plant against virus and provide useful guideline for the design of pathogen-derived amiRNAs.     

Csu-miR260转基因水稻插入序列的表达检测

本文以具有抗二化螟性状的Csu-miR260转基因水稻为试验材料, 采用TaqMan探针实时荧光定量PCR (TaqMan RT-qPCR)和茎环实时荧光定量PCR(stem-loop RT-qPCR)分别对Csu-miR260转基因抗虫水稻中Csu-miR260插入序列和剪切形成的amiR260s的表达情况进行了定量检测。发现Csu-miR260插入序列和剪切形成的20 nt和21 nt两种长度amiR260s在转基因抗虫水稻苗期的根、茎、叶中表达, 且无组织表达特异性。该试验解决了人工miRNA作物中amiRNA及前体检测引物的特异性问题, 为人工miRNA植物干扰序列的表达分析提供技术参考, 并为miRNA转基因作物遗传表达相关安全评价提供了数据参考。     

microRNA在猪中的研究进展

microRNA（miRNA）是一类在真核生物中广泛存在的非编码小RNA,通过与目标基因的3'端非编码序列结合,在转录后水平上调控基因的表达,参与许多生物学过程,包括细胞生长、分化、增殖和凋亡等。miRNA的生物合成始于细胞核,分为3个阶段,即pri-miRNA、pre-miRNA、成熟的miRNA分子。前两个阶段在细胞核中完成,第三阶段转移到细胞质中完成。miRNA具有在真核细胞中广泛存在、长度21~24 nt、序列具有高度保守性3个特点。研究表明,miRNA参与调控猪胚胎期肌肉发育、胚后期肌肉生长及肉质性状形成,猪前体脂肪细胞分化和脂质沉积,猪体的免疫应答,以及影响病原与机体的相互作用等过程。因此,了解更多的猪miRNA研究进展,可以加深对猪肌肉发育、脂肪代谢、疾病免疫等分子机制的理解,为生猪的健康养殖提供参考。     

猪CA5B基因的序列特征和表达分析

旨在通过分析猪CA5B(线粒体碳酸酐酶 VB,carbonic anhydrase 5b,mitochondria;CA5B/CA VB/Car5b)基因的序列特征和时空表达,解析CA5B对猪组织器官发育和代谢的影响.本研究采集6头240日龄贵州猪(公、母各3头)的心、肝、脾、肺、肾、皮下脂肪、背最长肌、睾丸、卵巢,采...     

miRNA调控畜禽剩余采食量的研究进展

剩余采食量（residual feed intake, RFI）是评价畜禽饲料利用率的重要指标。microRNA（miRNA）是一类通过转录后抑制基因表达的内源性非编码单链RNAs,长度约为22 nt,是基因表达的重要调控因子。miRNA与动物机体的采食及能量代谢密切相关,参与调控牛、猪、鸡等动物的RFI。针对近年来畜禽RFI的研究现状,特别是miRNA调控畜禽剩余采食量的研究进展进行总结,有助于揭示畜禽RFI性状的分子调控机理,为畜禽饲料利用率的改良与高效生产提供新思路,为优质畜禽的分子选育工作提供理论依据。     

microRNA与植物花发育调控的研究进展

microRNAs(miRNAs)是约21nt的非编码RNA,主要在转录后水平调节基因的活性。miRNAs通过与靶基因的互补位点结合从而降解靶基因mRNA或抑制其翻译。miRNAs 参与调控植物生长发育的多个方面,包括生长、开花、代谢、激素应答、生物与非生物胁迫。综述了miRNAs在植物花发育中的研究进展,以期为更好地了解miRNA在此过程中的作用机制,并应用于改良植物的农艺性状及培育优良品种奠定基础。     

沐川乌骨黑鸡肌内脂肪沉积相关microRNAs的筛查与鉴定

试验旨在挖掘高肌内脂肪（IMF）含量和低IMF含量鸡胸肌中差异表达的microRNA（miRNA）（DEM）,以期从miRNA角度探究鸡IMF沉积的分子调控机制。以120只沐川乌骨黑鸡为研究对象,采用高通量测序分别完成3个IMF含量极高（H组）和极低（L组）鸡胸肌组织的miRNA测序,测序结果经生物信息学分析,鉴定H和L组中差异表达的miRNAs,随机选择6个miRNAs进行实时荧光定量PCR验证。IMF含量测定结果表明,每只鸡平均100 g胸肌中IMF含量为4.08 g,H和L组IMF含量存在极显著差异（P<0.01）;6个测序文库结果表明,每个测序文库获得Clean reads均超过10 000 000条,Clean reads占Raw reads的百分比>93%,21~23 nt的small RNA（sRNA）数量占总sRNA的百分比>50%,其中22 nt长度的sRNA所占比例最高,平均GC碱基含量为48.95%,获得的测序数据可靠;6个测序文库中sRNA注释为rRNA、tRNA、snRNA、snoRNA以及重复序列的比例较低,分别为2.74%、1.85%、6.21%、1.58%、2.82%和2.81%;6个测序文库共鉴定已知miRNAs成熟体578个,miRNAs前体500个,鉴定差异表达的miRNAs 23个,其中H组中上调表达的miRNAs有16个,下调表达的miRNAs有7个;miRNA靶基因预测分析结果表明,23个差异表达miRNAs共预测靶基因628个;GO分析结果表明,共鉴定了30个显著富集GO条目,包括6个细胞成分、13个生物学过程和11个分子功能;KEGG富集分析表明,差异表达miRNA的靶基因显著富集于胰岛素信号通路、半乳糖代谢、脂肪细胞因子信号通路、鞘糖脂生物合成、糖酵解及淀粉和蔗糖代谢;实时荧光定量PCR结果表明,gga-miR-124a-3p、gga-let-7a-5p在H组中表达量极显著或显著高于L组（P<0.01;P<0.05）;gga-miR-19a-3p在H组中表达量极显著低于L组（P<0.01）,gga-miR-6553-3p和gga-miR-128-3p在H组中的表达量显著低于L组（P<0.05）,其表达趋势与测序结果一致。以上结果表明,差异表达的miRNA gga-miR-124a-3p、gga-let-7a-5p、gga-miR-6553-3p、gga-miR-128-3p和gga-miR-19a-3p参与脂肪生成,是鸡IMF含量调控重要的候选miRNAs,为阐释miRNA调控鸡IMF沉积分子机制提供理论基础。     

Differentially expressed microRNAs during solid endosperm development in coconut (Cocos nucifera L.)

MicroRNAs (miRNAs) are a class of 20–24 nt, endogenously expressed, non-coding RNAs that play important regulatory roles in plants and animals. To identify miRNAs potentially involved in tissue development and compound anabolism, we studied miRNA expression profiles in endosperm of coconut at different developmental stages. Based on the annotation in miRBase (release 10.1), we measured a total of 179 miRNAs in immature (95 expressed miRNAs) and mature tissues (176 expressed miRNAs) using microarrays, respectively. The comparative analyses on miRNA expression profiles between these two groups of tissues showed that 23 miRNAs were up-regulated and nine miRNAs were down-regulated in matured endosperm. We further confirmed the increased expression of four miRNAs and decreased expression of a miRNA in immature endosperm using real-time PCR. Moreover, we computationally predicted the target genes of 32 miRNAs with differential expression (p < 0.01), and identified the lowest-score targets of six miRNAs. Finally, we discussed the potential functional relevance of several differentially expressed miRNAs.