植物重要功能基因研究进展及其应用

随着越来越多植物全基因组测序的完成,植物基因研究的重点将逐渐从基因的发现转移到对基因功能的研究上来。近年来,植物基因功能的阐述日益深入,尤其是与作物产量和抗性相关的重要农艺性状调控机理的研究更加引人注目,一些具有应用价值的功能基因相继被鉴定并得到功能注释。该文综述植物功能基因研究领域近年来的主要进展,着重介绍具有应用前景的重要功能基因的研究。同时,对目前利用基因工程、分子标记辅助选择等于段改良作物的现状及其前景进行讨论。     

基因功能研究方法的新进展

随着生命科学的发展,研究领域的不断开拓,越来越多的未知新基因和基因的新功能被科学家们发现,研究这些未知新基因的功能和已知基因的新功能成为了极其重要的一项内容。本文对基因功能研究的最新方法进行了介绍。     

植物转座子及其在番茄功能基因组的应用

转座子作为插入突变原或分子标签被广泛应用于基因的分离和克隆,已成为发现新基因和基因功能分析的有效工具。该文综述了植物转座子及其在基因分离中的研究进展,并讨论其在番茄功能基因遗传资源、功能基因组分离等研究中的应用。     

RNA干扰文库及其在功能基因组学研究中的应用

随着人类基因组大规模测序的完成,下一步的挑战是了解每一个基因的功能 . RNA 干扰文库为大规模基因功能筛选提供了可能 . 虽然用于线虫等模式生物的 RNAi 文库,已经证明是大规模基因功能筛选的有效方法,但这些文库不能用于高等动物的细胞 . 自 2003 年以来,用于人的细胞和哺乳动物细胞的 RNAi 文库取得了突破,相继出现构建已知基因 RNAi 文库和构建随机 RNAi 文库的报道,并成功地应用于大规模基因功能的筛选 . RNAi 文库作为一种简单、高效、大规模、高通量的功能基因组学研究的工具,将在基因功能研究、发现新的药物靶基因、发现疾病相关基因等方面有广阔的应用前景 .     

T-DNA插入突变在植物功能基因组学中的应用

T-DNA插入突变在植物功能基因组学研究中发挥着重要作用,广泛应用于大规模植物基因功能分析,是分离新基因、研究基因功能的有效工具。我们简单介绍了T-DNA插入突变的原理,详细论述了3种T-DNA插入突变载体的应用,并综述了利用T-DNA插入突变克隆新基因的方法,同时指出了T-DNA插入突变存在的问题及其发展方向。     

白念珠菌基因功能研究的策略

白念珠菌是临床最常见的致病真菌,共有约6 100个基因,阐明其基因功能,尤其是致病性和耐药性相关基因的功能对发现抗真菌的新策略和新靶点有举足轻重的意义。白念珠菌基因功能研究的策略主要包括基因敲除和基因表达调控,近年来,白念珠菌基因功能研究的技术手段不断发展,本文就常用技术的发展进行综述,对相关技术存在的不足和发展前景也进行了分析。     

水稻基因组测序及基因功能的鉴定

水稻是重要的粮食作物。作为单子叶模式植物,水稻基因组的大规模测序具有巨大的理论价值和现实意义。目前已获得了籼稻“93—11”和粳稻“日本晴”高质量的基因组数据,这为在基因组水平上深入研究其生长、发育、抗病和高产等的遗传机理提供了便利,从而为进一步解决世界粮食危机提供了新的突破口和契机。随着水稻基因组计划的顺利结束,其研究重心也已由建立高分辨率的遗传、物理和转录图谱为主的结构基因组学转向基因功能的研究。结构基因组学研究获得的大量序列数据为揭示和开发功能基因开辟了广阔的前景。目前,利用图位克隆和电子克隆等方法已成功分离了多个水稻抗病、抗虫、抗逆境、抗倒伏、高产、优质等重要农艺性状相关的基因,对培育水稻新品种,促进农业的可持续发展意义重大。据估计,水稻至少拥有3．7万个非转座因子相关的蛋白编码基因。因此,完成全基因组序列测定后,重要基因功能的鉴定已成为当前基因组学研究的主要目标。反向遗传学、大规模基因功能表达谱分析和蛋白质组研究等策略已在研究水稻重要基因的功能方面发挥了重要作用。文章综述了水稻基因组测序及基因功能研究的现状,并就新基因发掘和基因功能注释的方法作了评述,期待为水稻遗传工程和育种实践提供参考。     

后基因组时代基因功能分析的策略

人类基因组计划的完成标志着生命科学已经开始迈进后基因组时代,大规模基因的功能研究正成为生命科学研究的热点。基因功能的分析方法日趋成熟和多样化,综述了基因敲除、反义技术、“dominantnegative”策略(“显性失活”策略)、基因诱导超表达、RNA干涉以及生物信息学等基因功能分析策略的特异性及其优缺点。     

基因组后研究策略

由于基因组计划的进展,数据库中积累了越来越多不知道功能的基因序列,分析这些基因的功能将成为基因组计划完成后的主要任务.文章介绍了几种大规模分析基因功能的策略及程序：基因插断、基因表达的系统分析、高密度cDNA杂交、蛋白组分析,并讨论了大规模基因功能分析的前景.     

酵母基因中断技术

酵母基因中断技术是研究酵母基因功能的重要手段,自80年代初诞生以来经历了不断的改进和发展.PCR介导的酵母基因中断技术,大大简化了操作,实现了酵母基因的精确缺失;酵母基因的多重中断技术,可在酵母内实现多个基因的中断;可进行大规模基因中断和功能分析的酵母基因中断技术,适应了在酵母全基因组测序完成的情况下进行功能基因组学研究的要求.酵母基因中断技术对人类基因功能研究也有很大启示作用.     

应用cDNA芯片分析79个新基因的人胚组织表达谱

大规模cDNA测序和生物信息学技术相结合,得到来自于商品化的人胚肾cDNA文库79个代表新基因的表达序列标签(EST).随后,采用高速度机械手制备这些cDNA的基因芯片,用于鉴定79个新基因的ESTs在20周、26周两个胚胎时期6种组织中的基因表达状况,以研究这些EST片段代表的新基因功能提供线索.通过芯片杂交及结果分析,得到同一个组织两个不同时相8个差异表达的基因,随后的RNA印迹分析的结果与芯片杂交的结果相一致.     

RFLP analysis of cry1 and cry2 genes of Bacillus thuringiensis isolates from India

The PCR-RFLP method has been useful for detection of known genes and identification of novel genes. In the present study, degenerate primers were designed from five groups of cry1 genes for PCR-RFLP analysis. Bacillus thuringiensis (Bt) isolates from different regions were evaluated for PCR amplification of various cry1 genes using newly designed primers and cry2 genes using reported primers. PCR analysis showed an abundance of cry1A genes and especially cry1Ac genes in isolates from all regions. RFLP analysis revealed the presence of multiple cry1A genes in isolates from central and southern regions. Unique digestion patterns of cry1A genes were observed in isolates from each region. Few of the isolates represented a digestion pattern of cry1A genes that did match to any of the known cry1A genes. RFLP analysis suggested an abundance of cry2Ab along with a novel cry2 gene in Bt isolates from different regions of India. Sequence analysis of the novel cry2 gene revealed 95% sequence identity to cry2Ab and cry2Ah genes. Phylogenetic analysis revealed that the novel cry2 gene could have diverged earlier than the other cry2 genes. Our results encourage finding of more diverse cry2 genes in Bt isolates. Rarefaction analysis was used to compare cry1A gene diversity in isolates from different soil types. It showed a higher degree of cry1A gene diversity in isolates from central region. In the present study, we propose the use of novel degenerate primers for cry1 genes and the PCR-RFLP method using a single enzyme to distinguish multiple cry1A and cry2 genes as well as identify novel genes.     

生物信息学在新基因全长cDNA序列分析及功能预测中的应用

差异表达基因的检测与分析已成为研究具有差异的生物学表型的常规策略.对通过实验所获得的差异基因片段进行生物信息学分析,主要包括基于国际互联网的序列相似性分析、片段重叠群分析和全长cDNA序列分析,以及如何构建局域网并采用本地服务器进行规模化的数据分析,从而为研究人员提供可参考的生物信息学数据分析方案.     

Evidence of a large novel gene pool associated with prokaryotic genomic islands

Microbial genes that are “novel” (no detectable homologs in other species) have become of increasing interest as environmental sampling suggests that there are many more such novel genes in yet-to-be-cultured microorganisms. By analyzing known microbial genomic islands and prophages, we developed criteria for systematic identification of putative genomic islands (clusters of genes of probable horizontal origin in a prokaryotic genome) in 63 prokaryotic genomes, and then characterized the distribution of novel genes and other features. All but a few of the genomes examined contained significantly higher proportions of novel genes in their predicted genomic islands compared with the rest of their genome (Paired t test = 4.43E-14 to 1.27E-18, depending on method). Moreover, the reverse observation (i.e., higher proportions of novel genes outside of islands) never reached statistical significance in any organism examined. We show that this higher proportion of novel genes in predicted genomic islands is not due to less accurate gene prediction in genomic island regions, but likely reflects a genuine increase in novel genes in these regions for both bacteria and archaea. This represents the first comprehensive analysis of novel genes in prokaryotic genomic islands and provides clues regarding the origin of novel genes. Our collective results imply that there are different gene pools associated with recently horizontally transmitted genomic regions versus regions that are primarily vertically inherited. Moreover, there are more novel genes within the gene pool associated with genomic islands. Since genomic islands are frequently associated with a particular microbial adaptation, such as antibiotic resistance, pathogen virulence, or metal resistance, this suggests that microbes may have access to a larger “arsenal” of novel genes for adaptation than previously thought.     

Identification of Pseudomonas syringae pv. tomato genes induced during infection of Arabidopsis thaliana

Phytopathogenic bacteria possess a large number of genes that allow them to grow and cause disease on plants. Many of these genes should be induced when the bacteria come in contact with plant tissue. We used a modified in vivo expression technology (IVET) approach to identify genes from the plant pathogen Pseudomonas syringae pv. tomato that are induced upon infection of Arabidopsis thaliana and isolated over 500 in planta-expressed (ipx) promoter fusions. Sequence analysis of 79 fusions revealed several known and potential virulence genes, including hrp/hrc, avr and coronatine biosynthetic genes. In addition, we identified metabolic genes presumably important for adaptation to growth in plant tissue, as well as several genes with unknown function that may encode novel virulence factors. Many ipx fusions, including several corresponding to novel genes, are dependent on HrpL, an alternative RNA polymerase sigma factor that regulates the expression of virulence genes. Expression analysis indicated that several ipx fusions are strongly induced upon inoculation into plant tissue. Disruption of one ipx gene, conserved effector locus (CEL) orf1, encoding a putative lytic murein transglycosylase, resulted in decreased virulence of P. syringae. Our results demonstrate that this screen can be used successfully to isolate genes that are induced in planta, including many novel genes potentially involved in pathogenesis.     

Novel estrogen-related genes and potential biomarkers of ovarian endometriosis identified by differential expression analysis

In the search for novel biomarkers of endometriosis, we selected 152 genes from the GeneLogic database based on results of genome-wide expression analysis of ovarian endometriosis, plus 20 genes related to estrogen metabolism and action. We then performed low-density array analysis of these 172 genes on 11 ovarian endometriosis samples and 9 control endometrium samples. Principal component analysis of the gene expression levels showed clear separation between the endometriosis and control groups. We identified 78 genes as differentially expressed. Based on Ingenuity pathway analysis, these differentially expressed genes were arranged into groups according to biological function. These analyses revealed that 32 differentially expressed genes are estrogen related, 23 of which have not been reported previously in connection with endometriosis. Functional annotation showed that 25 and 22 genes are associated with the biological terms "secreted" and "extracellular region", respectively. Differential expression of 4 out of 5 genes related to estrogen metabolism and action (ESR1, ESR2, PGR and BGN) was also confirmed by immunohistochemistry. Our study thus reveals differential expression of several genes that have not previously been associated with endometriosis and that encode potential novel biomarkers and drug targets.     

Genome-wide identification and evaluation of novel internal control genes for Q-PCR based transcript normalization in wheat

To accurately quantify gene expression using quantitative PCR amplification, it is vital that one or more ideal internal control genes are used to normalize the samples to be compared. Ideally, the expression level of those internal control genes should vary as little as possible between tissues, developmental stages and environmental conditions. In this study, 32 candidate genes for internal control were obtained from the analysis of nine independent experiments which included 333 Affymetrix GeneChip Wheat Genome arrays. Expression levels of the selected genes were then evaluated by quantitative real-time PCR with cDNA samples from different tissues, stages of development and environmental conditions. Finally, fifteen novel internal control genes were selected and their respective expression profiles were compared using NormFinder, geNorm, Pearson correlation coefficients and the twofold-change method. The novel internal control genes from this study were compared with thirteen traditional ones for their expression stability. It was observed that seven of the novel internal control genes were better than the traditional ones in expression stability under all the tested cDNA samples. Among the traditional internal control genes, the elongation factor 1-alpha exhibited strong expression stability, whereas the 18S rRNA, Alpha-tubulin, Actin and GAPDH genes had very poor expression stability in the range of wheat samples tested. Therefore, the use of the novel internal control genes for normalization should improve the accuracy and validity of gene expression analysis.