Establishment of TaqMan Real-time PCR Method for Detection of Duck Poxvirus

To establishment a TaqMan Real-time PCR method for detection of duck poxvirus (DPV),we cloned the P4b gene of DPV.The specific primers and probe were designed according to the nucleotide sequence of avipoxvirus available in GenBank.Recombinant plasmid pMD-DPV-P4b was employed as positive standard template for Real-time PCR.By optimization of reaction conditions,a TaqMan Real-time PCR method for detection of DPV was established.The results of specificity test proved this method had no cross-react with other waterfowl vial agents and poxviruses including avian influenza virus,duck flavivirus,duck hepatitis virus,Newcastle disease virus,duck entertitis virus,goose parvovirus,goatpox virus and fowlpox virus.The detection limit of the assay was 1.29×10² copies/μL of viral DNA,which was 100 times higher than that of the routine PCR.Reproducibility test showed that the CVs of intra assay and inter assay were both less than 2%.Above results supported that the assay was suitable for the detection of DPV very well.     

小麦果糖激酶基因TaFRK与TaPFT的互作分析

小麦赤霉病是全球性病害,严重威胁着中国的小麦粮食安全。目前已经明确的抗赤霉病位点有7个（ Fhb1~ Fhb7）。 其中 Fhb1位点是3BS染色体上稳定性和效应最大的抗扩展位点。 TaPFT是 Fhb1位点上的抗性基因之一,但其抗性机理并不清楚。本研究以TaFRK为诱饵蛋白通过酵母双杂交技术筛选小麦酵母双杂交文库,从而得到互作基因 TaFRK,其编码蛋白为果糖激酶。进一步通过构建酵母双杂交载体和双分子荧光互补载体分析 TaFRK与 TaPFT之间的互作关系,并通过qPCR分析 TaFRK受到禾谷镰刀菌孢子诱导后的表达模式。通过酵母双杂交试验发现,共转pGBKT7 - TaPFT和pGADT7 - TaFRK 的酵母菌株Y2HGlod在含有X-α-Gal和金担子素A（Aureobasidin A,AbA）的四缺培养基SD/-His-Leu-Trp-Ade上长出蓝色菌落,证明 TaFRK与 TaPFT在酵母中互作。通过双分子荧光互补试验发现,在共转YN- TaPFT和YC- TaFRK表达载体的烟草叶片中可观察到强烈的黄色荧光信号,表明 TaFRK与 TaPFT在植物细胞中能够发生相互作用。实时荧光定量PCR分析发现,禾谷镰刀菌孢子侵染抗性小麦后, TaFRK基因表达量升高,说明 TaFRK基因的表达受禾谷镰刀菌侵染诱导。本研究结果对进一步研究 TaPFT的赤霉病抗性机制具有一定的参考  价值。     

五指毛桃转录组及黄酮类化合物生物合成关键基因分析

五指毛桃含有香豆素类、黄酮类等化学成分,具有抗炎、抑菌、抗病毒和肿瘤的药用价值。但是,其转录组和功能基因组等分子水平的研究较少报道。本研究首次采用RNA-seq高通量测序技术对五指毛桃的根、茎和叶进行转录组测序,共获得104 335条unigenes,平均长度为578 bp,有62 142条unigenes被注释到Nr、GO、Swiss-Prot、KEGG和KOG等数据库中,根据同源序列比对发现五指毛桃与川桑的同源性最高。通过比较五指毛桃不同组织部位的转录组测序结果,发现Leaf_vs_Root、Root_vs_Stem、Stem_vs_Leaf分别有1363、624和1006个差异表达的基因在KEGG途径显著富集,主要富集在植物信号转导、植物MAPK信号通路、黄酮类生物合成等代谢途径。进一步分析参与植物黄酮类生物合成途径中的差异表达基因,发现有16个差异表达的基因参与该代谢途径,主要在叶和茎中高表达,运用RT-qPCR对黄酮类化合物生物合成途径中9个关键基因的表达量进行验证,发现定量结果与RNA-seq数据一致,大部分基因都在五指毛桃茎和叶片中高表达,因此五指毛桃幼苗黄酮类化合物主要积累在叶片和茎中,这与民间主要以根茎作为药用部位存在差异,主要原因可能是与五指毛桃生长年龄有关。以上的研究结果为五指毛桃分子生物学的研究奠定基础,并为其活性成分的生物合成调控、栽培技术等提供参考依据。     

喜树丛枝植原体的分子鉴定及TaqMan探针实时荧光定量PCR检测方法的建立

 为确定在云南文山地区喜树上发生的疑似丛枝病的病原种类及快速检测喜树丛枝病,本研究利用植原体16S rDNA基因通用引物P1/P7和R16F2n/R16R2对感病喜树总DNA进行常规PCR和巢式PCR扩增、克隆和测序,通过系统进化分析,明确了喜树丛枝植原体属于16SrXXXII组。然后根据喜树丛枝病植原体16S rDNA基因保守区域设计并合成特异性引物和TaqMan探针,制备了喜树丛枝病植原体标准质粒,确定了最优引物浓度和最佳探针浓度,制作的标准曲线有极好的线性关系,决定系数(R²)达到0.999,建立的实时荧光定量PCR检测方法能够特异性地检测喜树丛枝植原体。本研究首次明确了喜树丛枝植原体的分类地位,优化和建立了喜树丛枝植原体TaqMan探针qPCR检测方法,为快速检测喜树丛枝病植原体提供参考。     

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。     

仙台病毒核酸快速检测方法的建立和应用

仙台病毒(Sendai virus,SeV)是一种常见的可引起啮齿类动物呼吸道疾病的病原,感染迅速,并且隐性感染率高,一旦感染较难从鼠群中清除,从而影响动物健康。为满足对入境噬齿类实验动物和野生动物仙台病毒快速检测的需要,本研究针对SeV编码基质蛋白和融合糖蛋白基因的保守序列设计引物和荧光探针,建立了仙台病毒RT-PCR和Real-time RT-PCR检测方法。将建立的方法应用于仙台病毒感染鼠不同临床样品和组织培养物的检测,证实两种核酸检测方法具有良好的特异性、灵敏性和稳定性,适合应用于出入境口岸实验和野生噬齿类动物仙台病毒疫情的快速检测。     

四种油料作物中的细菌型PEPC基因的鉴定及在发育种子中的表达

鉴定和获取了四种油料作物（油菜、大豆、花生和芝麻）中的细菌型PEPC基因,分析了所编码蛋白的保守结构域（BOX I-IV）和蛋白作用功能位点。基因包括甘蓝型油菜的Bna10093361、Bna1009749和Bna10093360,大豆的Glyma10g34970.1, Glyma01g22840.1和Glyma02g14500.1,芝麻的SIN1018296和花生的AhPPC5。这8个基因含有19～21个内含子,内部插入一个约350～600bp的高度变异区,编码的蛋白在C端形成R/KNTG结构域,在N端缺乏磷酸化作用位点。在种子发育的不同时期,油菜中仅Bna10093360表达,但其表达量不到油菜BnActin表达量的0.1%;大豆中Glyma10g34970.1表达量最高（接近大豆GlymaActin的2%）,Glyma02g14500.1次之;花生AhPPC5表达量为花生AhActin的32%～175%,在种子不同发育时期表达量为早期>中期>晚期;芝麻SIN1018296表达量为芝麻SINActin的3%～18%,在种子发育时期的表达趋势和花生AhPPC5相似。8个基因种子中的表达模式差异明显,说明细菌型PEPC基因可能存在着广泛的功能分化。       

鸭呼肠孤病毒TaqMan与SYBR Green荧光定量检测方法的建立及比较

本研究旨在针对新型鸭呼肠孤病毒(NDRV)建立两种快速、敏感、特异的荧光定量PCR诊断方法并对其临床实用性进行比较.试验根据呼肠孤病毒S1基因序列设计合成1对特异性引物和1条MGB探针,分别建立TaqMan和SYBR GreenI两种荧光定量PCR检测方法,并对两种方法的特异性、灵敏性、重复性进行比较.结果 显示,两种...     

Profiling microRNA expression in bovine alveolar macrophages using RNA-seq

MicroRNAs (miRNAs) are important regulators of gene expression and are known to play a key role in regulating both adaptive and innate immunity. Bovine alveolar macrophages (BAMs) help maintain lung homeostasis and constitute the front line of host defense against several infectious respiratory diseases, such as bovine tuberculosis. Little is known, however, about the role miRNAs play in these cells. In this study, we used a high-throughput sequencing approach, RNA-seq, to determine the expression levels of known and novel miRNAs in unchallenged BAMs isolated from lung lavages of eight different healthy Holstein–Friesian male calves. Approximately 80 million sequence reads were generated from eight BAM miRNA Illumina sequencing libraries, and 80 miRNAs were identified as being expressed in BAMs at a threshold of at least 100 reads per million (RPM). The expression levels of miRNAs varied over a large dynamic range, with a few miRNAs expressed at very high levels (up to 800,000 RPM), and the majority lowly expressed. Notably, many of the most highly expressed miRNAs in BAMs have known roles in regulating immunity in other species (e.g. bta-let-7i, bta-miR-21, bta-miR-27, bta-miR-99b, bta-miR-146, bta-miR-147, bta-miR-155 and bta-miR-223). The most highly expressed miRNA in BAMs was miR-21, which has been shown to regulate the expression of antimicrobial peptides in Mycobacterium leprae-infected human monocytes. Furthermore, the predicted target genes of BAM-expressed miRNAs were found to be statistically enriched for roles in innate immunity. In addition to profiling the expression of known miRNAs, the RNA-seq data was also analysed to identify potentially novel bovine miRNAs. One putatively novel bovine miRNA was identified. To the best of our knowledge, this is the first RNA-seq study to profile miRNA expression in BAMs and provides an important reference dataset for investigating the regulatory roles miRNAs play in this important immune cell type.