SSH与cDNA芯片技术的联合及其在植物基因差异表达研究中的应用

抑制消减杂交技术(SSH)与cDNA芯片技术是新近发展起来的研究差异表达基因的两种非常有效的方法。近年来,将SSH和cDNA芯片技术结合使用,为分析组织细胞的已知基因表达差异提供了新的手段,也为克隆和鉴定新基因开辟了新的道路,是目前寻找差异表达基因的适用方法。鉴于此,对SSH和cDNA芯片技术的基本原理、两种方法结合使用的操作流程、克隆差异表达新基因的特点,以及在植物基因差异表达方面的应用进行了概述。     

猪蛔虫感染期幼虫抑制消减cDNA文库的构建

为筛选与线虫感染性相关的基因,本研究以猪蛔虫为对象,构建猪蛔虫感染期幼虫差异表达消减cDNA文库,为研究线虫期特异性发育的分子机制奠定基础。分别提取感染期幼虫和其它各期幼虫及成虫的总RNA,纯化mRNA后,采用Clontech公司PCR-selectTM试剂盒进行反转录合成cDNA并进行抑制消减杂交(SSH),构建猪蛔虫感染期幼虫差异表达的消减cDNA文库,并采用Southern斑点杂交进行消减效率的检测。随机从文库中抽取45个克隆进行测序及在线BLAST分析。试验结果表明,感染期幼虫差异表达的消减cDNA文库具有较强的特异性;在得到的41个表达序列标签(ESTs)中,有40个ESTs与已报道的基因有较高的相似性,主要代表猪蛔虫第三期幼虫基因和成虫头部基因,有1个cDNA片段可能代表新基因。猪蛔虫感染期幼虫差异表达消减cDNA文库的成功构建,为进一步研究幼虫发育差异表达基因的功能奠定了基础。     

检测猪流行性腹泻病毒、猪传染性胃肠炎病毒和猪轮状病毒的cDNA芯片的构建

猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪轮状病毒(PoRV)是三种常见的仔猪病毒性腹泻病原,作者拟构建可同时检测三种病原的cDNA基因芯片。分别根据PEDV基因(S、M)、TGEV基因(S、N)、PoRV基因(VP7、NSP4)的保守区设计引物扩增靶基因,进行PEDV-TGEV-PoRV的cDNA阵列设计,建立了扩增标记探针的多重PCR方法,并对所构建cDNA芯片的特异性、灵敏性、重复性进行了评价。确定了该cDNA芯片的阳性判断标准:SNR大于或等于2.0(或信号强度中位值大于等于1 500)。PEDV-TGEV-PoRV诊断cDNA芯片具有良好的特异性,该芯片不与猪繁殖与呼吸综合征病毒、猪瘟病毒、猪乙型脑炎病毒、猪伪狂犬病病毒发生交叉反应;芯片的最低检测质量浓度为20pg·μL-1;同一张芯片可重复使用至少7次。本研究为鉴别三种仔猪病毒性病的鉴别诊断提供了新的高通量检测技术。     

脑心肌炎病毒感染神经细胞的基因表达谱分析

脑心肌炎病毒(EMCV)可感染人和多种动物。为了探索EMCV对N2a细胞转录组的影响,揭示基因表达与致病性之间的关联,本研究用EMCV BD2毒株感染N2a细胞,收集感染后7 h的细胞样本,提取总RNA,使用NimbleGen杂交体系将标记好的ds-cDNA与小鼠基因表达谱芯片杂交,对芯片进行扫描,获得差异表达基因数据;应用生物信息学方法对筛查出的差异表达基因数据进行分析,筛选差异表达的免疫相关基因及信号通路;利用real-time FQ-PCR方法,对微阵列数据结果进行验证。结果表明,EMCV感染N2a细胞后共有21 143个差异表达基因;通路分析表明,差异表达的主要信号通路包括PI3K-Akt信号通路、MAPK信号通路、细胞因子间相互作用、趋化因子信号通路、TCR/RLR信号通路和细胞凋亡通路等;Real-time FQ-PCR检测的10个差异表达基因的变化情况与通过微阵列分析预测的变化一致。     

外源褪黑激素促进绒山羊皮肤毛囊相关基因表达差异的研究

旨在通过对非长绒期阿尔巴斯型绒山羊进行褪黑激素埋植,研究褪黑激素对羊绒周期性生长的调控,以期借此延长绒山羊毛囊的兴盛期,提高羊绒产量。将试验羊随机分成两组:埋植褪黑激素的试验组(T)和对照组(C),每组3只(试验组(TI、T2和T3)和对照组(C1、C2和C3)),采集绒山羊的皮肤组织进行组织切片,染色观察埋植褪黑激素对毛囊生长诱导作用。分别抽提两组样本总RNA,逆转录合成相应被标记的cDNA探针,采用Agilent绵羊的8×15K规格全基因组表达谱芯片进行杂交,筛选差异表达基因。利用实时荧光定量PCR技术进行验证。结果表明,从组织学分析可观察到埋植褪黑激素对毛囊生长具有明显的促进作用;芯片数据显示,筛选出差异表达的基因95个,其中61个表达上调、34个下调。GO分析基因数量分布情况:参与分子功能的基因占47.78%,参与生物学过程的基因占33.89%,组成细胞成分的基因占18.33%。与对照组的样本相比,埋植褪黑激素后相关基因的差异性表达涉及毛囊生长及周围皮肤附属物形态发生等生物学过程。这些差异性表达的基因为绒山羊毛囊生长及周期性调控的基因功能研究提供极有价值的参考。     

牦牛Fas和FasL基因克隆及其在主要组织器官中的表达

为了进一步理解Fas/FasL系统,本研究拟克隆牦牛Fas和FasL基因,并对2个基因在各组织器官中的表达进行检测。采用RT-PCR方法克隆牦牛Fas和FasL基因,并利用相关软件进行生物信息学分析,结合RTPCR方法及免疫组织化学方法对2个基因在牦牛主要组织器官中的表达进行检测。克隆得到了2 605bp的牦牛Fas基因cDNA和1 572bp的FasL基因cDNA序列,与黄牛的相应序列有很高的同源性(分别为99.1%和99.4%)。除了在心中只检测到Fas mRNA外,Fas和FasL mRNA共同表达于所有检测的组织中,且2个基因均在肝、脾、肾、淋巴结、胸腺和睾丸中强表达。免疫组化检测发现FasL蛋白存在于多种体细胞的胞浆中。结果表明,除了在淋巴器官和免疫赦免位点外,Fas和FasL还强表达于其他器官如肾和肝,表明Fas/FasL系统比以前所了解的要复杂。     

抑制性消减杂交技术在对虾基因克隆中的应用

抑制性消减杂交技术是一项筛选、分离未知差异表达基因的试验方法,该技术具有快速、简便、灵敏、特异、高效,所需起始样本量较少等优点。利用该技术构建对虾组织消减cDNA文库,鉴定差异表达相关基因及其表达特性。通过对对虾差异性表达的免疫相关基因、抗病基因及其他性状基因的了解,旨在为进一步研究对虾抗病机理奠定基础,为利用抗病基因进行抗病育种提供一个有效途径,为提高对虾的经济效益提供前景。     

奶牛乳腺组织中C4BPA基因的表达量差异分析

与奶牛乳脂相关的基因在奶牛乳腺组织中表达量的多少对奶牛的乳脂率起到关键的调控作用。C4BPA(complement component 4binding protein,alpha)基因是通过对高乳脂奶牛及低乳脂奶牛的乳腺组织进行转录组测序选择出的差异基因。为了研究C4BPA基因在奶牛乳脂合成代谢过程中的作用,本试验以中国荷斯坦奶牛为研究对象,利用乳成分分析仪检测15头中国荷斯坦奶牛的乳脂率,挑选出高乳脂奶牛与低乳脂奶牛各3头(P0.05),提取乳腺组织总RNA,反转录成cDNA,进行荧光定量PCR,检测该基因在奶牛乳腺组织中的表达量,并利用SPSS软件对不同奶牛乳腺组织中C4BPA基因的表达量进行差异分析。结果表明,C4BPA基因在不同奶牛乳腺组织中均有表达,且在高乳脂奶牛及低乳脂奶牛的乳腺组织中表达量差异显著(P0.05)。本试验为进一步对C4BPA基因生物学功能和作用机制的研究奠定基础。     

对虾白斑综合征病毒液相芯片快速检测方法的建立

本研究通过设计、合成并修饰基因特异性引物和寡核苷酸探针并标记生物素,利用该探针与荧光编码微球偶联后,与抽提的对虾白斑综合征病毒(White spot syndrome virus,WWSV)的PCR产物进行杂交反应,用液相芯片检测仪(Luminex200)检测荧光信号建立了WWSV快速液相芯片检测方法。该方法具有较好的特异性,偶联特异性探针的微球只与相应的病毒基因的PCR产物反应,而不与其他虾病病毒基因反应,可以检测到107的稀释度。本研究初步建立的WSSV液相芯片检测方法为进一步建立几种虾病同时快速检测奠定了基础。     

猪卵泡发育过程中差异表达miRNA的鉴定

本研究旨在探讨miRNAs对猪卵泡发育过程的影响。以卵泡期第4天杜洛克猪中等M1卵泡(直径3.0~4.9 mm)与M2卵泡(直径5.0~6.9 mm)为试验材料,通过miRNA芯片杂交检测筛选到在杜洛克母猪中等M1卵泡与M2卵泡之间的差异表达miRNAs,随机抽选若干个检测到的miRNA用Q-PCR的方法对芯片结果进行验证,并预测差异表达miRNA的靶基因,进行GO及KEGG分析。结果表明:芯片检测出347个猪miRNA的表达,筛选得到表达量高且差异大的34个miRNA;随机筛选出6个差异表达的miRNA经Q-PCR验证,与基因芯片结果一致;分析得到的靶基因涉及最多的为Jak-STAT、细胞因子及其受体的相互作用信号通路,且两个信号通路均与卵泡发育相关。结果提示:检测筛选得到的差异表达miRNA可能对猪的卵泡发育过程具有调控作用。     

家蚕第2白卵近等基因系差异表达基因的筛选

为了解与家蚕第2白卵(w-2)性状形成相关的差异表达基因信息,以家蚕正常型黑卵及其第2白卵近等基因系的转色期蚕卵为材料,构建抑制消减杂交(SSH)文库,筛选差异表达基因。对SSH文库中部分克隆的测序分析表明,该文库对差异表达基因的富集性较好。随机挑选SSH文库中的300个克隆制作家蚕cDNA芯片,对家蚕正常型黑卵及第2白卵近等基因系转色期蚕卵进行检测,获得11个差异表达基因。对这11个差异表达基因进行实时荧光定量RT-PCR验证分析,其结果与芯片数据分析结果趋势一致,在正常型黑卵与第2白卵近等基因系之间,这些基因的表达差异为0.1倍至数千倍。     

Microarray analysis of differentially expressed genes of primary tumors in the canine central nervous system

The pathophysiologic similarities of many human and canine cancers support the role of the domestic dog as a model for brain tumor research. Here we report the construction of a custom canine brain-specific cDNA microarray and the analysis of gene expression patterns of several different types of canine brain tumor. The microarray contained 4000 clones from a canine brain specific cDNA library including 2161 clones that matched known genes or expressed sequence tags (ESTs) and 25 cancer-related genes. Our study included 16 brain tumors (seven meningiomas, five glial tumors, two ependymomas, and two choroid plexus papillomas) from a variety of different dog breeds. We identified several genes previously found to be differentially expressed in human brain tumors. This suggests that human and canine brain tumors share a common pathogenesis. In addition, we also found differentially expressed genes unique to either meningiomas or the glial tumors. This report represents the first global gene expression analysis of different types of canine brain tumors by cDNA microarrays and might aid in the identification of potential candidate genes involved in tumor formation and progression.     

应用cDNA微阵列芯片技术筛选猪蛔虫性别差异表达基因的研究

采用cDNA微阵列芯片技术，从所构建的猪蛔虫雌、雄成虫cDNA消减文库分别挑取1044和1119个克隆，PCR扩增其插入片段，经纯化后点样于预先处理好的基片上（双点杂交），制备成cDNA微阵列芯片。将分别标记荧光素Cy3-dUTP和Cy5-dUTP的雌虫和雄虫cDNA探针，与制备好的cDNA芯片杂交（平行进行反标杂交试验）。根据每个点杂交后的Ratio值，筛选出双点杂交和正反标中都同时具有表达差异的基因克隆共1559个。将表达差异最明显的前831个克隆进行测序，获得720个有效序列，经生物信息学分析发现，雄虫特异表达的主要精于蛋白和雌虫特异表达的卵巢信息蛋白的基因序列多数与新杆属线虫存在同源性，有31个可能是新的ESTs。性别差异表达基因及其相关生物信息的获得为下一步研究基因功能奠定了基础。     

A mixed-model approach for the analysis of cDNA microarray gene expression data from extreme-performing pigs after infection with Actinobacillus pleuropneumoniae

We proposed a novel statistical approach for the analysis of cDNA experiments based on mixed-model methodology combined with mixtures of distributions. Our objective was to detect genes that may be involved in conferring heritable differences in susceptibility to common infections in intensive pig production. We employed a microarray expression profiling strategy and a mixed-model approach to the analysis of the expression data. A cDNA microarray of pig with 6,420 probes from immune tissues and cells was used to compare gene expression in peripheral blood leukocytes of two pigs showing extreme performance in their response to infection with Actinobacillus pleuropneumoniae. Principal components analyses were used to identify the two most extreme-performing pigs after infection (i.e., pigs whose measured responses to infection fell at the extremes). Blood samples and expression profiles from 0 to 24 h after infection were compared using a bivariate, mixed-model approach, in which the effect gene x immunological status interaction was treated as a random effect. Bayesian model-based clustering via mixtures of normal distributions of the resulting BLUP of the random interaction was approached and resulted in a list of 307 differentially expressed genes, of which 179 were down-regulated in the susceptible pig. The majority of the differentially expressed genes were derived from a cDNA library of leukocytes of A. pleuropneumoniae-challenged pigs that were subtracted against leukocytes before the challenge. These results provide evidence that the proposed statistical approach was useful in enhancing the knowledge of the mechanisms involved in the genetics of the immune response.     

A mixture model-based cluster analysis of DNA microarray gene expression data on Brahman and Brahman composite steers fed high-, medium-, and low-quality diets

The objective of this study is to explore aspects of the statistical analysis of gene expression response at the muscle tissue level to varying levels of energy and protein in the diet. Eleven Brahman and Brahman composite steers (weighing 302 +/- 9.8 kg, on average) were allocated randomly into high- (HIGH), medium- (MED), and low- (LOW) quality forage diets for 27 d. After this period, a biopsy of the longissimus dorsi muscle was taken from each animal and total RNA was extracted to generate the labeled target for microarray experimentation. These targets were hybridized to a complementary DNA (cDNA) microarray of 9,274 probes from cattle muscle and subcutaneous fat cDNA libraries. After edits, 151,904 expression intensity levels of 4,747 genes were analyzed. Emphasis was given to the choice of power transformation of the intensity channel readings and to the consistency of readings within each diet quality group. The statistical approach to isolate differentially expressed genes was based on model-based clustering via a mixture of normal distributions estimated through maximal likelihood. The base-2 logarithm was found to be the optimal power transformation to normalize gene intensity levels. A two-sample t-statistic was defined as a measure of possible differential expression. For each of the three diet contrasts, HIGH vs. LOW, HIGH vs. MED, and MED vs. LOW, three clusters were found, two of which contained more than 94% genes with almost no altered gene expression levels, whereas the third cluster contained the remaining genes with a differential expression. Results from the HIGH vs. LOW contrast identified 27 genes with a greater than 95% posterior probability of belonging to the cluster of differentially expressed genes.     

Gene discovery and expression profiling in porcine Peyer's patch

Peyer's patches of the intestinal mucosa are essential for host defense and immune regulation in the enteric system. To better understand molecular mechanisms of Peyer's patch function, we have screened for differentially expressed genes specific to Peyer's patch. cDNA libraries were created from normal Peyer's patch, immune stimulated Peyer's patch, and pooled cDNA subtracted with fibroblast RNA. From the subtracted library, 3687 expressed sequence tags (ESTs), representing 2414 unique nucleotide sequences, were isolated, identified by BLAST searches against public databases, and spotted onto a microarray for gene expression profiling. Approximately 30% of these ESTs BLAST to genes of unknown function and 20% have no known homology in the public databases (novel genes). Of the novel genes, 70% are expressed in normal immune tissues by microarray analysis, suggesting that at least 371 of the unidentified EST sequences from the subtracted library are novel porcine genes and can now be further characterized to determine their function in the porcine Peyer's patch. We surmise that the products of these genes participate in biochemical and cellular functions related to the unique immunological and gastroenterological functions of the small intestine. The BLAST and gene ontology information for each of the subtracted library EST sequences, the normal and immune stimulated libraries, and the microarray are all valuable resources that will facilitate further examination of the biological function of porcine Peyer's patch tissue.     

Effect of fescue toxicosis on hepatic gene expression in mice

Fescue toxicosis affects wild and domestic animals grazing fescue pasture infected with the endophytic fungus Neotyphodium coenophialum. Signs of fescue toxicosis include increased core body temperature and respiration rate and decreased milk yield and reproductive performance. Laboratory mice also exhibit symptoms of fescue toxicosis, as indicated by reduced growth rate and reproductive performance. Mice were used to study the effects of fescue toxicosis on hepatic gene expression. Twenty-seven mice were randomly allocated to a diet containing either 50% endophyte-infected (E+; 6 ppm ergovaline) or endophyte-free (E-) fescue seed for 2 wk under thermoneutral conditions. Liver genes differentially expressed due to fescue toxicosis were identified using DNA microarray. A 2-stage ANOVA of microarray data identified 36 differentially expressed genes between mice fed E+ and E- diets. Another analysis method, significance analysis of microarray, identified 9 genes as differentially expressed between treatment groups, and some genes overlapped with genes identified by ANOVA. Hierarchical clustering of 36 genes identified by ANOVA clearly separated the mice by diet, with 100% confidence as computed by bootstrap analysis. Expression of 11 genes was verified using quantitative real-time PCR. The E+ diet resulted in downregulation of genes involved in the sex-steroid metabolism pathway and genes involved in cholesterol and lipid metabolism. Genes coding for ribosomes and protein synthesis were upregulated by the E+ diet. Genes identified in the present analysis indicate some of the mechanisms by which fescue toxicosis occurs in animals.