Hela基因周期表达数据聚类及功能分析

目的:进一步了解与宫颈癌相关基因的功能以及作用关系.方法:根据Hela基因周期表达数据的时序特点,提出了KLCC聚类法.该方法以K-means法为平台,以基因问的局部相关系数(Local Correlative Coefficient,LCC)为相关性测度,并对K-means法进行了相应的改进,根据基因间的相关关系,分批对基因聚类.结果:在对Hela基因周期表达数据的聚类分析中,得到9个显著的功能类,其中有3个与肿瘤紧密相关,具有优于当前常用算法的性能.结论:KLCC法能够有效地识别Hela基因周期表达数据中的局部相关和异步相关,并对其进行功能显著的聚类,为宫颈癌的基因治疗提供参考和依据.     

马氏珠母贝Sox11基因的克隆及时序表达模式分析

为探究Sox(SRY-related HMG-box genes)基因家族在马氏珠母贝个体发育及性别分化中的作用, 研究首先利用兼并引物从马氏珠母贝基因组中克隆到一个HMG框(high mobility group box), 利用RACE-PCR技术从SMART cDNA文库中克隆到一个Sox基因的cDNA全长, 通过Clustal X和MEGA 4软件对该序列进行比对分析并构建系统进化树; 通过荧光定量PCR技术对该基因在不同组织及发育不同时期性腺中的表达情况进行分析。结果显示, 马氏珠母贝该Sox基因的cDNA全长为1579 bp, 其中开放阅读框(ORF)为1008 bp, 编码336个氨基酸, 5'非编码区为126 bp, 3'非编码区为445 bp。同源性分析表明, 马氏珠母贝Sox基因与太平洋牡蛎Sox11基因的同源性(Identity)最高, 为80%, 故命名为pmSox11; 系统进化树分析也显示pmSox11与太平洋牡蛎Sox11基因的亲缘关系最近。荧光定量PCR分析组织表达特异性显示, pmSox11在马氏珠母贝神经节分布较多的组织如外套膜、鳃、足、消化盲囊等大量表达, 在神经节相对较少的闭壳肌和卵巢中表达量较少; 时序表达图谱显示, pmSox11在3月龄幼贝性腺和1年龄发育早期精巢中表达量最高, 在2年龄成熟精巢和2年龄性转换性腺中表达量降低, 而在2年龄卵巢中表达量最低。研究表明, pmSox11基因可能在马氏珠母贝早期神经系统发育和性别发育的调控方面起到重要作用。     

鸡PPARs基因组织表达特性的研究

以8周龄Arber Acres(AA)肉鸡为研究材料,采用RT-PCR和Northern blot方法对鸡PPAR基因组织表达特点进行了研究。RT-PCR半定量检测显示,在检测的10种组织中除胸部肌肉组织外,PPAR-α基因在其他9种组织中都表达,其中较高表达于脑、肺脏、肾脏、心脏和小肠,中等程度表达于胃、肝脏和脂肪,较低表达于脾脏。PPAR-γ基因除了在肝脏和肌肉中没有检测到外,在其他8种组织都有表达,其中高表达于脂肪,其次是脑和肾脏,低量表达于脾脏、心脏、肺脏、胃和小肠。Northern blot检测显示,PPAR-α基因在心脏、肝脏、肾脏和胃这4种组织表达,其中在肝脏杂交信号最强;PPAR-γ基因只在脂肪和肾脏表达,其中在脂肪组织有强的杂交信号。以上结果表明,鸡PPARS基因的组织表达特点同啮齿动物和人基本一致,但也有其自身的特殊性,即PPAR-α基因不在肌肉组织中表达,PPAR-γ基因在肾脏中有高表达。PPAR基因与鸡的多种组织的生长和发育有关。     

不同病原菌刺激下三疣梭子蟹主要组织LGBP基因的时序表达

脂多糖-β-1,3-葡聚糖结合蛋白（lipopolysaccharide-β-1,3-glucan binding protein,LGBP）作为一种模式识别受体能够识别革兰氏阴性菌和真菌的细胞壁多糖成分脂多糖和β-1,3-葡聚糖,在甲壳动物的免疫系统中占有重要地位,为了解LGBP在蟹类动物抗菌免疫应答中的作用,采用SYBR Green I实时荧光定量PCR方法,对分别经溶藻弧菌,酵母菌,溶-酵混合菌、金黄色葡萄球菌刺激6 h、12 h、24 h、48 h的三疣梭子蟹血细胞、肝胰腺、肌肉3种组织LGBP基因的相对表达量进行了检测。结果显示,LGBP基因在3种组织中的时序表达量总体趋势基本一致,呈现＂上升-下降-再上升-下降＂的趋势,各实验组在实验6 h时表达量均达到了峰值;不同病原菌刺激下LGBP基因在梭子蟹肝胰腺和血细胞中的表达量明显高于肌肉,且溶-酵混合菌刺激组织LGBP的表达量最高,酵母菌和溶藻弧菌刺激组次之,金黄色葡萄球菌组最低,说明LGBP主要参与蟹类抵抗革兰氏阴性菌和真菌侵入的免疫防御反应,但对革兰氏阳性菌也有一定的作用。     

水稻花药绒毡层特异表达基因RA39的克隆与表达特性分析

利用cDNA减法杂交,差异杂交筛选和RACE等技术。从水稻（Orza sativaL.ssp.japonica)中克隆了一个新的绒毡层特异性cDNA,其编码基因被命名为RA39。该cDNA长1013bp。编码由298个氨基酸残基组成的多肽RA39是一个单拷贝基因。在绒毡层细胞中特异性表达。在小孢子母细胞减数分裂期的绒毡层细胞中有较高的表达活性。用PSORT和PPSEARCH软件进行的结构分析揭示出RA39蛋白的N端是一个由17个氨基酸残基组成的信号肽,该蛋白包含一个跨膜区和一个胞质尾区两个主要结构域以及多个蛋白激酶的磷酸化位点。     

ApoE基因缺失幼龄小鼠动脉粥样硬化相关基因的时序表达研究

利用RT-PCR技术检测apoE基因缺失(apoE-/-小鼠在1、2和3月龄3个年龄段主动脉以及14天、1、2和3月龄4个年龄段肝脏中动脉粥样硬化(atherosclerosis,AS)重要相关基因的时序表达特点,并通过血清牛化检测结合主动脉根部病理形态学特征的分析,探讨AS重要相关基因时序表达特点与AS早期病变的关系.在主动脉检测的9条AS相关基因中,apoE-/-与野生型(WT)小鼠相比,1、2和3月龄时IL-1β的mRNA表达水平均显著上调.1月龄时VCAM-1、IKB和TGF-β,2月龄时PDGF-α和CD36,3月龄时TNF-α和MM2的mRNA表达水平均显著上调,其他年龄段无显著变化.在肝脏检测的2条AS相关基因中,C反应蛋白(CRP)的mRNA表达水平在14天到2月龄时无显著变化,3月龄时显著上调.NF-kB在各年龄段apoE-/-小鼠主动脉和肝脏中的基因表达量与同龄WT小鼠相比均无显著差异.不同年龄段的apoE-/-小鼠血清TC、TG和LDL-C水平均明显高于同龄WT小鼠.2月龄apoE-/-小鼠主动脉内膜出现散在的脂质沉积,并随年龄增长逐渐加重.上述结果显示,时序表达上调的AS重要相关基因构成了以NF-kB为核心的复杂调控网络,它们之间相互作用,共同参与慢性炎症过程,在apoE-/-小鼠AS的早期发生发展中发挥重要作用.     

gfP基因甘蓝质体表达载体构建及原核表达分析

以甘蓝自交系‘03097’为试材,PCR扩增并克隆了甘蓝质体的trnI-trnA基因区段,利用该基因区段作为定点整合外源基因的同源重组片段,构建了甘蓝质体定点转化载体,并进行了原核表达分析。结果表明,所扩增的基因区段大小为2.7 kb。进行序列分析后,经酶切、连接和转化,构建成含Prrn-gfp-aadA-TpsbA表达盒的质体表达载体,酶切鉴定表明,所构建载体符合预期设计;gfp基因能够在质体特异性启动子Prrn及终止子TpsbA的调控下高水平表达,表达量约占总可溶性蛋白的41.0%,包涵体占菌体蛋白的38.0%。该载体的构建为后期甘蓝质体转化体系的建立和其它功能基因导入甘蓝质体进行性状改良奠定了基础。     

籽粒苋中PPDK基因的克隆及表达特性分析

为寻找能提高植物光合效率的基因资源,以高光效植物籽粒苋(Amaranthus hypochondriacus L.)为试材,利用同源克隆和RACE技术克隆了丙酮酸磷酸二激酶(Pyruvate orthophosphate dikinase, PPDK)基因,基因cDNA全长为3 224 bp,其中5′非翻译区为71 bp,阅读框为2 868 bp,3′非翻译区为285 bp,推导的蛋白质为956个氨基酸,分子量约106 kDa。序列分析表明,克隆的基因含有PPDK基因的功能结构域。表达模式分析显示克隆的PPDK基因在绿色组织中特异表达,为PPDK基因的长转录本,初步确定已克隆得到为籽粒苋中的PPDK基因,将其命名为AhPPDK。     

猪脂肪组织发育过程中Wnt/β-catenin信号通路相关基因及脂肪转录因子的时序表达

为研究Wnt/b-catenin信号通路对猪脂肪组织发育的影响, 并探讨其可能的作用机制, 以1~120日龄长白猪脂肪组织为试验材料, 采用SQ RT-PCR法检测Wnt信号通路中相关基因β-catenin、GSK3β、Fz1及主要的脂肪转录因子PPARγ、C/EBPα 和分化早期标志基因LPL mRNA的表达变化; 石蜡切片免疫组化方法定性检测β-catenin在脂肪组织发育中的时序表达变化。SQ RT-PCR结果显示, β-catenin mRNA在猪出生第1天有高水平表达, 随着个体日龄的增长, 其表达量逐渐降低, 60日龄后维持在一个较低水平。GSK3β和Fz1 mRNA的表达量也伴随脂肪组织的发育而逐渐降低。而LPL、PPARγ和C/EBPα的 mRNA表达量随着个体日龄的增长而逐渐升高, 60日龄后仍保持高水平的表达。石蜡切片免疫组化结果显示, 随着脂肪组织的发育, β-catenin蛋白的表达也随之降低, 并且β-catenin蛋白的表达部位逐渐由细胞核和细胞质共表达变为只在细胞质中表达。上述基因表达的时序变化规律提示, β-catenin在维持前体脂肪细胞的未分化状态, 抑制脂肪组织发育中具有重要作用, 其作用机制可能是通过对脂肪细胞转录因子PPARγ、C/EBPα及分化早期标志基因LPL mRNA的调控来进行的。     

Adaptive thresholds to detect differentially expressed genes in microarray data

To detect changes in gene expression data from microarrays, a fixed threshold for fold difference is used widely. However, it is not always guaranteed that a threshold value which is appropriate for highly expressed genes is suitable for lowly expressed genes. In this study, aiming at detecting truly differentially expressed genes from a wide expression range, we proposed an adaptive threshold method (AT). The adaptive thresholds, which have different values for different expression levels, are calculated based on two measurements under the same condition. The sensitivity, specificity and false discovery rate (FDR) of AT were investigated by simulations. The sensitivity and specificity under various noise conditions were greater than 89.7% and 99.32%, respectively. The FDR was smaller than 0.27. These results demonstrated the reliability of the method.     

Finding edging genes from microarray data

MOTIVATION: A set of genes and their gene expression levels are used to classify disease and normal tissues. Due to the massive number of genes in microarray, there are a large number of edges to divide different classes of genes in microarray space. The edging genes (EGs) can be co-regulated genes, they can also be on the same pathway or deregulated by the same non-coding genes, such as siRNA or miRNA. Every gene in EGs is vital for identifying a tissue's class. The changing in one EG's gene expression may cause a tissue alteration from normal to disease and vice versa. Finding EGs is of biological importance. In this work, we propose an algorithm to effectively find these EGs. RESULT: We tested our algorithm with five microarray datasets. The results are compared with the border-based algorithm which was used to find gene groups and subsequently divide different classes of tissues. Our algorithm finds a significantly larger amount of EGs than does the border-based algorithm. As our algorithm prunes irrelevant patterns at earlier stages, time and space complexities are much less prevalent than in the border-based algorithm. AVAILABILITY: The algorithm proposed is implemented in C++ on Linux platform. The EGs in five microarray datasets are calculated. The preprocessed datasets and the discovered EGs are available at http://www3.it.deakin.edu.au/~phoebe/microarray.html.     

Identifying differentially expressed genes in human acute leukemia and mouse brain microarray datasets utilizing QTModel

One of the essential issues in microarray data analysis is to identify differentially expressed genes (DEGs) under different experimental treatments. In this article, a statistical procedure was proposed to identify the DEGs for gene expression data with or without missing observations from microarray experiment with one- or two-treatment factors. An F statistic based on Henderson method III was constructed to test the significance of differential expression for each gene under different treatment(s) levels. The cutoff P value was adjusted to control the experimental-wise false discovery rate. A human acute leukemia dataset corrected from 38 leukemia patients was reanalyzed by the proposed method. In comparison to the results from significant analysis of microarray (SAM) and microarray analysis of variance (MAANOVA), it was indicated that the proposed method has similar performance with MAANOVA for data with one-treatment factor, but MAANOVA cannot directly handle missing data. In addition, a mouse brain dataset collected from six brain regions of two inbred strains (two-treatment factors) was reanalyzed to identify genes with distinct regional-specific expression patterns. The results showed that the proposed method could identify more distinct regional-specific expression patterns than the previous analysis of the same dataset. Moreover, a computer program was developed and incorporated in the software QTModel, which is freely available at .     

Rapid divergence in expression between duplicate genes inferred from microarray data

For more than 30 years, expression divergence has been considered as a major reason for retaining duplicated genes in a genome, but how often and how fast duplicate genes diverge in expression has not been studied at the genomic level. Using yeast microarray data, we show that expression divergence between duplicate genes is significantly correlated with their synonymous divergence (K_S) and also with their nonsynonymous divergence (K_A) if K_A ≤ 0.3. Thus, expression divergence increases with evolutionary time, and K_A is initially coupled with expression divergence. More interestingly, a large proportion of duplicate genes have diverged quickly in expression and the vast majority of gene pairs eventually become divergent in expression. Indeed, more than 40% of gene pairs show expression divergence even when K_S is ≤ 0.10, and this proportion becomes >80% for K_S > 1.5. Only a small fraction of ancient gene pairs do not show expression divergence.     

鸡胚公母性腺差异表达基因的基因芯片杂交筛选

采用Affymetrix公司鸡基因组芯片对9日龄鸡胚公母性腺总RNA进行了芯片杂交, 并对基因表达谱进行了分析。统计结果显示, 9日龄母鸡性腺表达基因数19 368个, 公鸡性腺表达基因数19 493个; 公母性腺绝对差异表达基因,即公鸡性腺表达而母鸡性腺不表达基因145个, 母鸡性腺表达而公鸡性腺不表达基因189个。绝对差异表达基因功能分类结果显示, 参与细胞组成、细胞加工和分子结合基因占多数, 部分基因参与细胞器组成、代谢加工、生物学调控以及催化反应和细胞信号转导等。值得注意的是, 本研究发现了一些已经报道同性别决定和分化有一定关联的基因, 如ASW、CHD1和SOX9等, 同时也发现了一些未知其同性腺分化和发育有关联的基因和编码假想蛋白的表达序列。进一步分析这些基因和表达序列的生物学功能和表达模式, 将对鸟类性别决定和分化机制的了解提供有益参考。     

Identifying differentially expressed genes from microarray experiments via statistic synthesis

MOTIVATION: A common objective of microarray experiments is the detection of differential gene expression between samples obtained under different conditions. The task of identifying differentially expressed genes consists of two aspects: ranking and selection. Numerous statistics have been proposed to rank genes in order of evidence for differential expression. However, no one statistic is universally optimal and there is seldom any basis or guidance that can direct toward a particular statistic of choice. RESULTS: Our new approach, which addresses both ranking and selection of differentially expressed genes, integrates differing statistics via a distance synthesis scheme. Using a set of (Affymetrix) spike-in datasets, in which differentially expressed genes are known, we demonstrate that our method compares favorably with the best individual statistics, while achieving robustness properties lacked by the individual statistics. We further evaluate performance on one other microarray study.     

Identification of genes related to Parkinson's disease using expressed sequence tags.

In a search for novel target genes related to Parkinson's disease (PD), two full-length cDNA libraries were constructed from a human normal substantia nigra (SN) and a PD patient's SN. An analysis of the gene expression profiles between them was done using the expressed sequence tags (ESTs) frequency. Data for the differently expressed genes were verified by quantitative real-time RT-PCR, immunohistochemical analysis and a cell death assay. Among the 76 genes identified with a significant difference (P > 0.9), 21 upregulated genes and 13 downregulated genes were confirmed to be differentially expressed in human PD tissues and/or in an MPTP-treated mice model by quantitative real-time RT-PCR. Among those genes, an immunohistochemical analysis using an MPTP mice model for alpha-tubulin including TUBA3 and TUBA6 showed that the protein levels are downregulated, as well as the RNA levels. In addition, MBP, PBP and GNAS were confirmed to accelerate cell death activity, whereas SPP1 and TUBA3 to retard this process. Using an analysis of ESTs frequency, it was possible to identify a large number of genes related to human PD. These new genes, MBP, PBP, GNAS, SPP1 and TUBA3 in particular, represent potential biomarkers for PD and could serve as useful targets for elucidating the molecular mechanisms associated with PD.