最短路径法在水稻ABA和环境胁迫条件下基因应答网络研究中的应用

目前微阵列数据分析方法都基于具有相似表达模式的基因可能具有相近的生物学功能这一假设,而实际上参与同一生物学功能的基因,在表达时间和空间上是有关联的,而并非表现为相似模式。利用水稻cDNA微阵列,对水稻在ABA及干旱、寒冷和高盐胁迫条件下的基因表达进行了研究。选取环境胁迫和ABA应答的相关基因,采用最短路径法(shortest path),利用自行编制的计算软件,在表达模式不直接相关的基因之间构建最短路径。研究表明,通过分析这些基因的表达数据,可以发现它们在功能上的关联性,并对未知基因的功能预测进行了探索,为构建水稻在ABA和环境胁迫条件下的分子应答网络奠定了基础。     

水稻Rop基因OsRac5表达特性

Rop在植物生长、发育、免疫及环境信号应答等多种生物学过程中具有重要作用。已有研究显示水稻Rop基因OsRac5可能与育性控制有关,但是该基因的表达特性,以及非生物胁迫和植物生长物质对其表达的影响尚不清楚。本文采用qRT-PCR技术检测了OsRac5在水稻生长发育过程中、非生物胁迫以及植物生长物质处理条件下的表达特性,结果显示OsRac5在水稻生长发育过程中在多种组织广泛表达,尤其在根和雌雄蕊形成期的幼穗中高表达;干旱、高盐和低温等非生物胁迫均能诱导OsRac5表达;ABA、GAs、6-BA等植物生长物质能上调OsRac5基因表达,提示该基因与水稻幼穗发育、抗逆性及细胞生长等过程相关。     

番茄AT-hook基因家族的鉴定及胁迫条件下的表达分析

AT-hook蛋白家族在植物生长发育、器官构建及逆境胁迫和激素信号应答中发挥重要作用。本研究在番茄基因组范围内,利用生物信息学方法对番茄AT-hook基因家族的成员、分布、结构和功能进行分析。结果表明,番茄AT-hook家族包含32个成员,分为3种类型,其中类型Ⅰ含有13个成员;遗传进化分析表明番茄AT-hook基因成员与拟南芥家族基因具有相似分类。利用实时荧光定量PCR对番茄32个基因开展组织表达分析,结果表明AT-hook基因具有表达差异,主要在根和花中表达较高。氧化胁迫分析结果表明,32个基因受ABA、SA、盐、高温和低温诱导表达,其中部分基因显著上调或下调表达,很可能参与了番茄逆境胁迫条件下的防御应答反应。本研究结果将为番茄AT-hook家族基因的深入研究提供依据,为进一步解析番茄AT-hook基因的功能奠定基础。     

水稻OsMPK15的cDNA克隆和转录水平分析

MAPK基因参与水稻的生物与非生物胁迫应答及生长发育过程,通过克隆水稻OsMPK15并初步研究其表达特性,为后续功能研究奠定基础。采用RT-PCR技术从日本晴水稻的根组织中克隆OsMPK15的cDNA编码区,并采用实时荧光定量PCR(qRT-PCR)技术分析OsMPK15在不同组织中以及不同非生物胁迫下的表达特性。结果表明,从日本晴水稻根组织中克隆的OsMPK15的cDNA编码区序列与生物信息学预测的结果一致,OsMPK15的第13-304位为丝氨酸/苏氨酸激酶结构域,属于水稻MAPK家族E组。qRT-PCR结果表明,该基因在水稻幼穗期各组织表达存在差异,主要在叶和叶鞘中表达。干旱和盐胁迫均可诱导OsMPK15在水稻幼苗根中表达量的上调,但表达模式具有不同的规律。同时,OsMPK15对3种胁迫相关激素ABA、JA和SA处理均产生应答,其中以ABA诱导OsMPK15表达上调幅度最大。     

玉米钼辅助因子硫酸化酶基因启动子的克隆与功能验证

为克服组成型启动子启动外源基因过量表达引起的诸多问题,同源克隆(Mo-molybdopterin cofactor sulfurase)基因（ABA3）的启动子(ABA3s)序列,并用PlantCARE软件分析其非生物逆境应答元件, 实时定量PCR检测ABA3基因在非生物逆境诱导下的差异表达后。然后,用该启动子构建启动GUS（β-glucuronidase）基因的表达载体, 基因枪法转化玉米愈伤组织。经组织化学染色法检测其表达后, 在高渗、高盐、低温胁迫处理及ABA诱导下检测GUS酶荧光值与荧光素酶(内参)发光值的比值(GUS/LUC), 以此评价ABA3s启动子在非生物逆境胁迫下的启动活性。结果表明, ABA3基因在模拟干旱、低温、高温、高盐胁迫及ABA、乙稀诱导下差异表达, 说明该基因的启动子(ABA3s)具有非生物逆境诱导活性。序列分析表明, ABA3s启动子全长777 bp, 含有ARE、HSE、MBS、TGA、Circadian等多种非生物逆境胁迫应答元件。用ABA3s启动GUS基因构建的表达载体转化的玉米愈伤组织, 响应干旱、低温、高温、高盐胁迫等多种非生物逆境胁迫, 及ABA和乙稀诱导, GUS检测呈阳性。在8%甘露醇高渗条件下, GUS/LUC比值比空白对照高6倍。上述结果表明, ABA3s启动子具有非生物逆境诱导特性, 经进一步验证其功能后, 可用于玉米抗逆转基因研究。     

脱落酸对水稻抽穗开花期高温胁迫的诱抗效应

为探究脱落酸(ABA)对水稻(Oryza sativa)抽穗开花期高温胁迫的诱抗效应, 以江西省主推水稻品种黄华占为材料, 于孕穗期用蒸馏水、ABA溶液(10、50和100 µmol∙L^-1)、氟啶酮(FLU)和原花青素(PC) 6种溶液进行叶面喷施, 然后移入对照(CK)和高温胁迫(HS)环境处理8天, 考查籽粒活性氧(ROS)积累、抗氧化防御能力、产量构成及相关基因的表达。结果表明, 高温胁迫下, 水稻的穗长、穗重、结实率、千粒重和产量与超氧阴离子和过氧化氢含量呈显著负相关。高温胁迫下, 喷施ABA显著上调了ABA应答和抗氧化防御基因的表达, 籽粒中活性氧含量下降了8.24%-31.35%; 喷施ABA显著增加了水稻的穗长、穗重、结实率和千粒重, 显著上调了产量形成基因的表达, 增产12.73%-20.77%。高温胁迫下, 喷施FLU可抑制ABA的生物合成, 导致ROS过量积累和水稻减产; 喷施抗氧化剂PC则抑制ROS过量积累, 使产量增加。以上结果表明, 高温胁迫下, 孕穗期喷施ABA不仅能够激发ABA信号通路, 而且上调抗氧化防御能力和产量形成基因的表达, 进而提高水稻在抽穗开花期的耐热性, 达到增产目的。     

陆地棉GhPYR1基因的克隆和功能分析

植物激素脱落酸(Abscisic acid,ABA)在植物应对干旱、盐碱等逆境胁迫以及植物种子萌发、根伸长、芽休眠等阶段发挥重要作用。PYR/PYL/RCAR蛋白家族是ABA受体,与ABA结合后能够启动ABA信号传导通路,诱导ABA应答基因的表达。利用电子克隆和RT-PCR技术从陆地棉中克隆了Gh PYR1基因,其编码的Gh PYR1蛋白与拟南芥中At PYR1蛋白相似度为73%。将Gh PYR1蛋白序列与拟南芥14个PYR/PYL/RCAR家族成员蛋白序列进行比对并构建进化树,发现它与拟南芥PYR/PYL/RCAR蛋白亚家族III亲缘关系最近。过表达Gh PYR1基因的T3代拟南芥在外源ABA处理下,其种子萌发和初期根生长均滞后于野生型,表现出对ABA更加敏感;高盐和干旱胁迫对转基因种子的萌发抑制更强烈,但苗期胁迫处理下转基因拟南芥的长势却明显优于野生型;同时在外源ABA诱导条件下ABA应答基因RD29A、RAB18的表达量较野生型有明显提高。以上结果说明Gh PYR1基因编码的蛋白是ABA的受体,过表达该基因能够提高植物对ABA的敏感性和增强应对逆境胁迫的能力。     

通过cDNA微阵列鉴定水稻(Oryza sativa L.) 盐胁迫应答基因

利用水稻耐盐品种兰胜构建了一个高质量的cDNA文库以鉴定水稻盐胁迫应答基因, 从cDNA文库中提取约15000个质粒, 并用Biomek 2000 高密度点阵系统或手工操作, 将这些质粒点于尼龙膜上. 通过这种方法鉴定了30个盐应答基因, 对其中的12个基因通过Northern杂交进行表达分析, 确证了cDNA微阵列的杂交结果. 30个基因中, 18个基因受盐诱导, 另外12个基因受盐抑制, 其中27个在GenBank数据库中有同源序列. 根据功能, 这些基因大致可以分为5类：光合作用相关基因、物质运输相关基因、代谢相关基因、耐逆相关基因以及其他未分类的基因. 研究结果表明, 盐胁迫影响了植物生长发育的多个方面, 其中有些基因可能在植物体的耐盐过程中具有重要作用.     

SRWD: a novel WD40 protein subfamily regulated by salt stress in rice (OryzasativaL.)

By analysis with microarray data, we found that a gene encoding a novel protein containing five WD40 repeats, was regulated by salt stress in rice and named as SRWD1 (Salt responsive WD40 protein 1). By database searching, additional four SRWD1-like genes (SRWD2-SRWD5) were found in rice genome, and these five SRWD genes formed a novel WD40 subfamily. Phylogenetic analysis showed that plant SRWD proteins divided into four groups. The significant functional divergences during SRWD evolution were found. The tissue-specific and salt responsive expression profiling for SRWD genes was investigated based on microarray data. It was found that all five SRWD genes in rice were regulated by salt stress. Further, we found that SRWD1 was regulated with different patterns by salt stress in two rice cultivars responding differently to salt stress. Our study correlates WD40 proteins with salt stress in plants and provides fundamental information for the further investigation of plant SRWD proteins.     

Identification and functional analysis of light-responsive unique genes and gene family members in rice

Functional redundancy limits detailed analysis of genes in many organisms. Here, we report a method to efficiently overcome this obstacle by combining gene expression data with analysis of gene-indexed mutants. Using a rice NSF45K oligo-microarray to compare 2-week-old light- and dark-grown rice leaf tissue, we identified 365 genes that showed significant 8-fold or greater induction in the light relative to dark conditions. We then screened collections of rice T-DNA insertional mutants to identify rice lines with mutations in the strongly light-induced genes. From this analysis, we identified 74 different lines comprising two independent mutant lines for each of 37 light-induced genes. This list was further refined by mining gene expression data to exclude genes that had potential functional redundancy due to co-expressed family members (12 genes) and genes that had inconsistent light responses across other publicly available microarray datasets (five genes). We next characterized the phenotypes of rice lines carrying mutations in ten of the remaining candidate genes and then carried out co-expression analysis associated with these genes. This analysis effectively provided candidate functions for two genes of previously unknown function and for one gene not directly linked to the tested biochemical pathways. These data demonstrate the efficiency of combining gene family-based expression profiles with analyses of insertional mutants to identify novel genes and their functions, even among members of multi-gene families.     

Comprehensive expression analysis of rice phospholipase D gene family during abiotic stresses and development

Phospholipase D is one of the crucial enzymes involved in lipid mediated signaling, triggered during various developmental and physiological processes. Different members of PLD gene family have been known to be induced under different abiotic stresses and during developmental processes in various plant species. In this report, we are presenting a detailed microarray based expression analysis and expression profiles of entire set of PLD genes in rice genome, under three abiotic stresses (salt, cold and drought) and different developmental stages (3-vegetative stages and 11-reproductive stages). Seven and nine PLD genes were identified, which were expressed differentially under abiotic stresses and during reproductive developmental stages, respectively. PLD genes, which were expressed significantly under abiotic stresses exhibited an overlapping expression pattern and were also differentially expressed during developmental stages. Moreover, expression pattern for a set of stress induced genes was validated by real time PCR and it supported the microarray expression data. These findings emphasize the role of PLDs in abiotic stress signaling and development in rice. In addition, expression profiling for duplicated PLD genes revealed a functional divergence between the duplicated genes and signify the role of gene duplication in the evolution of this gene family in rice. This expressional study will provide an important platform in future for the functional characterization of PLDs in crop plants.     

Clustering of gene expression data using a local shape-based similarity measure

MOTIVATION: Microarray technology enables the study of gene expression in large scale. The application of methods for data analysis then allows for grouping genes that show a similar expression profile and that are thus likely to be co-regulated. A relationship among genes at the biological level often presents itself by locally similar and potentially time-shifted patterns in their expression profiles. RESULTS: Here, we propose a new method (CLARITY; Clustering with Local shApe-based similaRITY) for the analysis of microarray time course experiments that uses a local shape-based similarity measure based on Spearman rank correlation. This measure does not require a normalization of the expression data and is comparably robust towards noise. It is also able to detect similar and even time-shifted sub-profiles. To this end, we implemented an approach motivated by the BLAST algorithm for sequence alignment.We used CLARITY to cluster the times series of gene expression data during the mitotic cell cycle of the yeast Saccharomyces cerevisiae. The obtained clusters were related to the MIPS functional classification to assess their biological significance. We found that several clusters were significantly enriched with genes that share similar or related functions.     

Differential quantitative regulation of specific gene groups and pathways under drought stress in rice

Abiotic stresses like drought are detrimental for growth and development and lead to loss in crop production. To be able to adapt and survive under such adverse conditions, synchronous regulation of a rather large number of genes is required. Here, we have used a bioinformatics approach to identify gene groups and associated pathways from microarray and RNA-seq experiments that are restricted in their gene expression amplitude within fold change intervals (FCI) under drought stress conditions. We find that the expression of genes as functional groups is coordinated quantitatively, in a fold change specific manner, and differs among three rice cultivars distinct in their drought stress response. By networking these groups and further categorization into components like ubiquitin proteasome system, we identify relatively less studied E2 ubiquitin conjugating enzyme coding genes as an important constituent of differential drought stress response in rice. By extending this approach to find hexamer DNA motifs in the upstream promoter regions of genes within the FCIs under stress, we find that genes with strong to very strong or a moderate expression under stress are coordinated through cis-regulatory motifs. Few of these, such as TSO1, L-Box, PE1, GT binding site, ABRE/G-box or AP2/ERF binding site can be candidate cis-regulatory motifs to coordinate fold change limited gene expression under drought stress. This work thus provides an insight into a quantitative regulation of gene expression under drought stress in rice and a useful resource for designing approaches towards coordinating the expression of identified candidate genes under stress in order to achieve drought tolerance in rice.     

Effective similarity measures for expression profiles

It is commonly accepted that genes with similar expression profiles are functionally related. However, there are many ways one can measure the similarity of expression profiles, and it is not clear a priori what is the most effective one. Moreover, so far no clear distinction has been made as for the type of the functional link between genes as suggested by microarray data. Similarly expressed genes can be part of the same complex as interacting partners; they can participate in the same pathway without interacting directly; they can perform similar functions; or they can simply have similar regulatory sequences. Here we conduct a study of the notion of functional link as implied from expression data. We analyze different similarity measures of gene expression profiles and assess their usefulness and robustness in detecting biological relationships by comparing the similarity scores with results obtained from databases of interacting proteins, promoter signals and cellular pathways, as well as through sequence comparisons. We also introduce variations on similarity measures that are based on statistical analysis and better discriminate genes which are functionally nearby and faraway. Our tools can be used to assess other similarity measures for expression profiles, and are accessible at biozon.org/tools/expression/     

Web Tools for Rice Transcriptome Analyses

Gene expression databases provide profiling data for the expression of thousands of genes to researchers worldwide. Oligonucleotide microarray technology is a useful tool that has been employed to produce gene expression profiles in most species. In rice, there are five genome-wide DNA microarray platforms: NSF 45K, BGI/Yale 60K, Affymetrix, Agilent Rice 44K, and NimbleGen 390K. Presently, more than 1,700 hybridizations of microarray gene expression data are available from public microarray depositing databases such as NCBI gene expression omnibus and Arrayexpress at EBI. More processing or reformatting of public gene expression data is required for further applications or analyses. Web-based databases for expression meta-analyses are useful for guiding researchers in designing relevant research schemes. In this review, we summarize various databases for expression meta-analyses of rice genes and web tools for further applications, such as the development of co-expression network or functional gene network.     

The Ortholog Conjecture Is Untestable by the Current Gene Ontology but Is Supported by RNA Sequencing Data

The ortholog conjecture posits that orthologous genes are functionally more similar than paralogous genes. This conjecture is a cornerstone of phylogenomics and is used daily by both computational and experimental biologists in predicting, interpreting, and understanding gene functions. A recent study, however, challenged the ortholog conjecture on the basis of experimentally derived Gene Ontology (GO) annotations and microarray gene expression data in human and mouse. It instead proposed that the functional similarity of homologous genes is primarily determined by the cellular context in which the genes act, explaining why a greater functional similarity of (within-species) paralogs than (between-species) orthologs was observed. Here we show that GO-based functional similarity between human and mouse orthologs, relative to that between paralogs, has been increasing in the last five years. Further, compared with paralogs, orthologs are less likely to be included in the same study, causing an underestimation in their functional similarity. A close examination of functional studies of homologs with identical protein sequences reveals experimental biases, annotation errors, and homology-based functional inferences that are labeled in GO as experimental. These problems and the temporary nature of the GO-based finding make the current GO inappropriate for testing the ortholog conjecture. RNA sequencing (RNA-Seq) is known to be superior to microarray for comparing the expressions of different genes or in different species. Our analysis of a large RNA-Seq dataset of multiple tissues from eight mammals and the chicken shows that the expression similarity between orthologs is significantly higher than that between within-species paralogs, supporting the ortholog conjecture and refuting the cellular context hypothesis for gene expression. We conclude that the ortholog conjecture remains largely valid to the extent that it has been tested, but further scrutiny using more and better functional data is needed.