青枯菌诱导的花生基因表达谱SSH分析

以抗青枯病花生种质‘J4’和‘中花6号’、感青枯病花生品种‘中花12号’为材料,用强产青枯菌毒菌株(Ralstonia solanacearum)对其根系分别接种,采用抑制差减杂交(SSH)技术检测花生根系应答侵染的基因表达谱变化,并对文库中差异基因进行Real-time PCR分析。结果表明:经菌液PCR检测对挑选出的1 036阳性克隆片段进行测序及片段整合分析,获得162条花生基因,有功能注释的基因58条,其中44条基因参与了细胞结构(6%)、信号转导(12%)、抗病防御(5%)、转录调控(12%)等生理过程。用Real-time PCR技术对7个基因在‘中花6号’和‘中花12号’中的表达模式分析结果表明,6个基因在青枯菌侵染早期在抗病材料‘中花6号’中呈上调表达,可能与青枯病抗性直接相关。     

MicroRNA-499 Expression Distinctively Correlates to Target Genes sox6 and rod1 Profiles to Resolve the Skeletal Muscle Phenotype in Nile Tilapia

A class of small non-coding RNAs, the microRNAs (miRNAs), has been shown to be essential for the regulation of specific cell pathways, including skeletal muscle development, maintenance and homeostasis in vertebrates. However, the relative contribution of miRNAs for determining the red and white muscle cell phenotypes is far from being fully comprehended. To better characterize the role of miRNA in skeletal muscle cell biology, we investigated muscle-specific miRNA (myomiR) signatures in Nile tilapia fish. Quantitative (RT-qPCR) and spatial (FISH) expression analyses revealed a highly differential expression (forty-four-fold) of miR-499 in red skeletal muscle compared to white skeletal muscle, whereas the remaining known myomiRs were equally expressed in both muscle cell types. Detailed examination of the miR-499 targets through bioinformatics led us to the sox6 and rod1 genes, which had low expression in red muscle cells according to RT-qPCR, FISH, and protein immunofluorescence profiling experiments. Interestingly, we verified that the high expression of miR-499 perfectly correlates with a low expression of sox6 and rod1 target genes, as verified by a distinctive predominance of mRNA destabilization and protein translational decay to these genes, respectively. Through a genome-wide comparative analysis of SOX6 and ROD1 protein domains and through an in silico gene regulatory network, we also demonstrate that both proteins are essentially similar in vertebrate genomes, suggesting their gene regulatory network may also be widely conserved. Overall, our data shed light on the potential regulation of targets by miR-499 associated with the slow-twitch muscle fiber type phenotype. Additionally the results provide novel insights into the evolutionary dynamics of miRNA and target genes enrolled in a putative constrained molecular pathway in the skeletal muscle cells of vertebrates.     

人受精促进肽受体基因（TCP11b）的剪接、克隆和差别表达

运用同源比较和PCR法 ,从人睾丸组织中分离了人受精促进肽受体TCP11基因的一个新的剪切体TCP11b ,它编码 5 0 3个氨基酸的蛋白质 ,与TCP11a相比 ,在基因组的 5′端存在复杂的外显子剪接现象。运用荧光原位杂交 (FISH)方法 ,显示该基因定位到人染色体 6p2 1。Northern杂交及多组织RT PCR的结果显示该转录本在正常睾丸中表达 ,而其他组织、无精症患者及胎儿睾丸组织中未见该基因的表达。该结果结合mTcp 11功能的提示 ,TCP11b这种转录本对精子发生和人受精过程可能起重要作用。     

选择性剪接在低氧应答中的调控作用

细胞为适应低氧环境,其相关基因的表达方式发生了改变,其中选择性剪接在低氧应答调控过程中起到了重要的作用。低氧诱导因子介导的低氧应答信号通路在机体适应低氧环境过程中起到了十分重要的作用,低氧诱导因子剪接体通过此通路调控红细胞生成、血管生成、糖酵解等过程。而抑制性PAS蛋白质、脯氨酸羟化酶、促血管生长因子、芳香羟受体核转运蛋白剪接体则通过其它通路进行调控。选择性剪接不仅在低氧应答中起重要作用,而且与阿尔茨海默病、动脉粥样硬化、癌症等常见人类疾病相关。     

Integrating Circadian Activity and Gene Expression Profiles to Predict Chronotoxicity of Drosophila suzukii Response to Insecticides

Native to Southeast Asia, Drosophila suzukii (Matsumura) is a recent invader that infests intact ripe and ripening fruit, leading to significant crop losses in the U.S., Canada, and Europe. Since current D. suzukii management strategies rely heavily on insecticide usage and insecticide detoxification gene expression is under circadian regulation in the closely related Drosophila melanogaster, we set out to determine if integrative analysis of daily activity patterns and detoxification gene expression can predict chronotoxicity of D. suzukii to insecticides. Locomotor assays were performed under conditions that approximate a typical summer or winter day in Watsonville, California, where D. suzukii was first detected in North America. As expected, daily activity patterns of D. suzukii appeared quite different between ‘summer’ and ‘winter’ conditions due to differences in photoperiod and temperature. In the ‘summer’, D. suzukii assumed a more bimodal activity pattern, with maximum activity occurring at dawn and dusk. In the ‘winter’, activity was unimodal and restricted to the warmest part of the circadian cycle. Expression analysis of six detoxification genes and acute contact bioassays were performed at multiple circadian times, but only in conditions approximating Watsonville summer, the cropping season, when most insecticide applications occur. Five of the genes tested exhibited rhythmic expression, with the majority showing peak expression at dawn (ZT0, 6am). We observed significant differences in the chronotoxicity of D. suzukii towards malathion, with highest susceptibility at ZT0 (6am), corresponding to peak expression of cytochrome P450s that may be involved in bioactivation of malathion. High activity levels were not found to correlate with high insecticide susceptibility as initially hypothesized. Chronobiology and chronotoxicity of D. suzukii provide valuable insights for monitoring and control efforts, because insect activity as well as insecticide timing and efficacy are crucial considerations for pest management. However, field research is necessary for extrapolation to agricultural settings.     

Pre-Slaughter Stress Affects Ryanodine Receptor Protein Gene Expression and the Water-Holding Capacity in Fillets of the Nile Tilapia

Current study evaluated the effect of pre-slaughter stress on serum cortisol levels, pH, colorimetry, water-holding capacity (WHC) and gene expression of ryanodine receptors (RyR1 and RyR3) in the Nile tilapia. A 3x4 factorial scheme experiment was conducted comprising three densities (100, 200, 400 kg/m³) with four transportation times (60, 120, 180, and 240 minutes).Transportation times alone reduced cortisol levels up to 180 minutes, followed by increased WHC and mRNA expression, RyR1 and RyR3 (200 kg/m³ density). No effect of density x transportation time interacted on the evaluated parameters. Results provided the first evidence that pre-slaughter stress affected ryanodine gene expression receptors and, consequently, the water-holding capacity in tilapia fillets.     

Integrative Meta-Analysis of Differential Gene Expression in Acute Myeloid Leukemia

Background

Acute myeloid leukemia (AML) is a heterogeneous disease with an overall poor prognosis. Gene expression profiling studies of patients with AML has provided key insights into disease pathogenesis while exposing potential diagnostic and prognostic markers and therapeutic targets. A systematic comparison of the large body of gene expression profiling studies in AML has the potential to test the extensibility of conclusions based on single studies and provide further insights into AML.

Methodology/Principal Findings

In this study, we systematically compared 25 published reports of gene expression profiling in AML. There were a total of 4,918 reported genes of which one third were reported in more than one study. We found that only a minority of reported prognostically-associated genes (9.6%) were replicated in at least one other study. In a combined analysis, we comprehensively identified both gene sets and functional gene categories and pathways that exhibited significant differential regulation in distinct prognostic categories, including many previously unreported associations.

Conclusions/Significance

We developed a novel approach for granular, cross-study analysis of gene-by-gene data and their relationships with established prognostic features and patient outcome. We identified many robust novel prognostic molecular features in AML that were undetected in prior studies, and which provide insights into AML pathogenesis with potential diagnostic, prognostic, and therapeutic implications. Our database and integrative analysis are available online (http://gat.stamlab.org).     

Gene Expression Analysis Suggests Temporal Differential Response to Aluminum in Coffea arabica Cultivars

Aluminum (Al) is a limiting factor of crop yields on acidic soils. Ion aluminum (Al³⁺) acts primarily in plant root system retarding its growth and development, leading to the reduction of lateral roots number, and consequently the decrease of vegetal production. Most of coffee producing areas are located in acidic soils, which have Al³⁺ contents enough to damage plant development. Despite the advances in the understanding of physiological and genetic mechanisms of Al tolerance/susceptibility, few are known about Al ion action in coffee plants. This report describes the expression analysis of genes related to aluminum stress in germinating seeds of two cultivars of C. arabica (Catuaí Amarelo IAC 62 and Icatu Vermelho IAC 4045) when challenged with Al³⁺. In silico analyses of Brazilian Coffee Genome Project (BCGP) database were used to select genes previously found to be related with Al-stress. The expression profile of these genes in Catuaí and Icatu was evaluated through Quantitative PCR (qPCR). Based on our data, we suggest that both analyzed cultivars displays mechanisms of resistance or exclusion, which occurs outside the cell excluding Al³⁺ assimilation, and mechanisms of tolerance that occurs inside the cell after Al³⁺ absorption. The major difference is the timing of activation of each mechanism. While Catuaí tends to use resistance mechanisms in early stages of stress, Icatu uses tolerance strategies. In late stages, both cultivars seem to display tolerance mechanisms, but Icatu also displays Al-exclusion strategy.     

盐地碱蓬过氧化氢酶基因的克隆及盐胁迫下的表达分析

过氧化氢酶是清除H2O2的重要酶类.从400 mmol/L NaCl处理的盐地碱蓬(Suaeda salsa(L.)Pall)地上部分的cDNA文库中克隆了两个编码过氧化氢酶的cDNA(Sscat1和Sscat2),其中Sscat1(1.7kb)是一个全长cDNA克隆,编码一个492个氨基酸的开放阅读框架,而Sscat2(1.1kb)是一个cDNA片段.据编码Sscat1 3＇端的287个氨基酸的cDNA序列与Sscat2的cDNA序列进行的BLAST同源性分析表明,Sscat1和Sscat2在核苷酸水平的一致性为71.9%,在氨基酸水平上的一致性为75%.Southem杂交表明,Sscat1在盐地碱蓬基因组中为多拷贝基因,Sscat2则为一个单拷贝基因.Northern杂交结果表明在盐胁迫条件下Sscat1和Sscat2的表达存在差异:400 mmol/L NaCl处理48h的盐地碱蓬根中的Sscat1和Sscat2 mRNA水平比对照显著提高,但是在叶中仅Sscatl受盐诱导表达.不同盐处理时间下的表达分析也证实,在盐地碱蓬叶中仅Sscat1受盐诱导表达.这说明Sscat1和Sscat2在盐地碱蓬中是差异调控的.生理分析表明过氧化氢酶的活性在盐胁迫条件下显著提高.     

盐地碱蓬过氧化氢酶基因的克隆及盐胁迫下的表达分析

过氧化氢酶是清除H2O2的重要酶类,从400mmol/LNaCl处理的盐地碱蓬（Suaeda salsa(L.)Pall)地上部分的cDNA文库中克隆了两个编码过氧化氢酶的cDNA(Sscat1和Sscat2)。其中Sscatl(1.7kb)是一个全长cDNA克隆,编码一个492个氨基酸的开放阅读框架。而Sscat2(1.1kb)是一个cDNA片段。据编码Sscatl3′端的287个氨基酸的cDNA序列与Sscat2的cDNA序列进行的BLAST同源性分析表明,Sscat1和Sscat2在核苷酸水平的一致性则为一个单拷贝基因。Northern杂交结果表明在盐胁迫条件下Sscat1和Sscat2的表达存在差异;400mmol/LNaCl处理48h的盐地碱蓬根中的Sscat1和Sscat2mRNA水平比对照显著提高,但是在叶中仅Sscat1受盐诱导表达,不同盐处理时间下的表达分析也证实。在盐地碱蓬叶中仅Sscat1受盐诱导表达。这说明Sscat1和Sscat2在盐地碱蓬中是差异调控的,生理分析表明过氧化氢酶的活性在盐胁迫条件下显著提高。     

滋养层细胞侵袭相关基因表达谱分析

分离收集正常妊娠第8～12周的细胞滋养层细胞和绒毛外滋养层细胞,提取细胞总RNA,制备cRNA探针并与AffymetrixU133plus2.0基因芯片进行杂交,获得正常细胞滋养层细胞和绒毛外滋养层细胞基因表达谱芯片。经计算机分析共筛选到1318个差异表达基因,其中上调基因813个,下调505个。所有差异表达基因按GeneOntoloty功能分类标准进行了功能检索。为胚胎发育早期绒毛外滋养层细胞侵袭的基因调控机制的研究提供了实验基础。     

Response Prediction to Neoadjuvant Chemotherapy: Comparison between Pre-Therapeutic Gene Expression Profiles and In Vitro Chemosensitivity Assay

Although the use of (neo-)adjuvant chemotherapy in breast cancer patients has resulted in improved outcome, not all patients benefit equally. We have evaluated the utility of an in vitro chemosensitivity assay in predicting response to neoadjuvant chemotherapy. Pre-therapeutic biopsies were obtained from 30 breast cancer patients assigned to neoadjuvant epirubicin 75 mg/m2 and docetaxel 75 mg/m2 (Epi/Doc) in a prospectively randomized clinical trial. Biopsies were subjected to a standardized ATP-based Epi/Doc chemosensitivity assay, and to gene expression profiling. Patients then received 3 cycles of chemotherapy, and response was evaluated by changes in tumor diameter and Ki67 expression. The efficacy of Epi/Doc in vitro was correlated with differential changes in tumor cell proliferation in response to Epi/Doc in vivo (p = 0.0011; r = 0.73670, Spearmańs rho), but did not predict for changes in tumor size. While a pre-therapeutic gene expression signature identified tumors with a clinical response to Epi/Doc, no such signature could be found for tumors that responded to Epi/Doc in vitro, or tumors in which Epi/Doc exerted an antiproliferative effect in vivo. This is the first prospective clinical trial to demonstrate the utility of a standardized in vitro chemosensitivity assay in predicting the individual biological response to chemotherapy in breast cancer.     

Differential Response of Obese Gene Expression from Fasting in Bovine Adipose Tissues

To understand the molecular mechanism for intramuscular fat deposition, the expression of the obese gene was examined in response to fasting. Food deprivation for 48 h induced a decrease in the level of obese mRNA in pooled adipose tissues (abdominal, perirenal, subcutaneous, intermuscular and intramuscular). The expression of obese mRNA was examined for individual adipose tissue from several fat depots. It was highly expressed in perirenal adipose tissue, but fasting did not affect its expression level in this tissue. Moderate levels were detected in subcutaneous and intermuscular adipose tissues, and a fasting-induced decrease in obese mRNA was apparent in these tissues. The expression level of the obese gene in intramuscular adipose tissue was very low and did not respond to fasting.     

Annotation of Differential Gene Expression in Small Yellow Follicles of a Broiler-Type Strain of Taiwan Country Chickens in Response to Acute Heat Stress

This study investigated global gene expression in the small yellow follicles (6–8 mm diameter) of broiler-type B strain Taiwan country chickens (TCCs) in response to acute heat stress. Twelve 30-wk-old TCC hens were divided into four groups: control hens maintained at 25°C and hens subjected to 38°C acute heat stress for 2 h without recovery (H2R0), with 2-h recovery (H2R2), and with 6-h recovery (H2R6). Small yellow follicles were collected for RNA isolation and microarray analysis at the end of each time point. Results showed that 69, 51, and 76 genes were upregulated and 58, 15, 56 genes were downregulated after heat treatment of H2R0, H2R2, and H2R6, respectively, using a cutoff value of two-fold or higher. Gene ontology analysis revealed that these differentially expressed genes are associated with the biological processes of cell communication, developmental process, protein metabolic process, immune system process, and response to stimuli. Upregulation of heat shock protein 25, interleukin 6, metallopeptidase 1, and metalloproteinase 13, and downregulation of type II alpha 1 collagen, discoidin domain receptor tyrosine kinase 2, and Kruppel-like factor 2 suggested that acute heat stress induces proteolytic disintegration of the structural matrix and inflamed damage and adaptive responses of gene expression in the follicle cells. These suggestions were validated through gene expression, using quantitative real-time polymerase chain reaction. Functional annotation clarified that interleukin 6-related pathways play a critical role in regulating acute heat stress responses in the small yellow follicles of TCC hens.