Progress in TILLING as a tool for functional genomics and improvement of crops

Food security is a global concern and substantial yield increases in crops are required to feed the growing world population. Mutagenesis is an important tool in crop improvement and is free of the regulatory restrictions imposed on genetically modified organisms. Targeting Induced Local Lesions in Genomes(TILLING), which combines traditional chemical mutagenesis with high‐throughput genome‐wide screening for point mutations in desired genes, offers a powerful way to create novel mutant alleles for both functional genomics and improvement of crops. TILLING is generally applicable to genomes whether small or large, diploid or evenallohexaploid, and shows great potential to address the major challenge of linking sequence information to the function of genes and to modulate key traits for plant breeding. TILLING has been successfully applied in many crop species and recent progress in TILLING is summarized below, especially on the developments in mutation detection technology, application of TILLING in gene functional studies and crop breeding. The potential of TILLING/EcoTILLING for functional genetics and crop improvement is also discussed. Furthermore, a small‐scale forward strategy including backcross and selfing was conducted to release the potential mutant phenotypes masked in M2(or M3) plants.     

TILLING moves beyond functional genomics into crop improvement

Transgenic methods have been successfully applied to trait improvement in a number of crops. However, reverse genetics studies by transgenic means are not practical in many commercially important crops, hampering investigations into gene function and the development of novel and improved cultivars. A nontransgenic method for reverse genetics called Targeting Induced Local Lesions IN Genomes (TILLING) has been developed as a method for inducing and identifying novel genetic variation, and has been demonstrated in the model plant, Arabidopsis thaliana. Recently, TILLING has been extended to the improvement of crop plants and shows great promise as a general method for both functional genomics and modulation of key traits in diverse crops.     

TILLING技术在作物突变研究中的应用现状与前景

定向诱导基因组局部突变(Targeting Induced Local Lesions In Genomes,TILLING)技术将化学诱变与高通量突变检测技术相结合,可高效、快速地从突变群体中鉴定出目标基因突变位点。本文在概述TILLING技术应用于水稻、小麦、玉米、大豆等作物突变研究现状基础上,重点综述了TILLING分析群体构建与突变位点检测方法的技术改进与发展,探讨了TILLING技术目前存在的问题与前景。     

TILLING in extremis

Targeting induced local lesions in genomes (TILLING), initially a functional genomics tool in model plants, has been extended to many plant species and become of paramount importance to reverse genetics in crops species. Because it is readily applicable to most plants, it remains a dominant non-transgenic method for obtaining mutations in known genes. The process has seen many technological changes over the last 10 years; a major recent change has been the application of next-generation sequencing (NGS) to the process, which permits multiplexing of gene targets and genomes. NGS will ultimately lead to TILLING becoming an in silico procedure. We review here the history and technology in brief, but focus more importantly on recent developments in polyploids, vegetatively propagated crops and the future of TILLING for plant breeding.     

TILLING技术在植物功能基因组及育种中的应用

随着拟南芥、水稻等模式植物基因组测序计划的全面完成,数据库中大量的DNA序列需要进行功能注释,而用传统的正向遗传学进行基因克隆和近年来发展的反向遗传学（如插入突变、反义RNA、RNAi等技术）方法已不能适应基因组学的发展需求,因此,研发大规模、高通量的基因功能分析方法成为当务之急。TILLING技术（Targeting induced local lesions in genomes）就是在基因组生物学大背景下出现的一种全新的反向遗传学技术。TILLING技术的基本步骤是通过化学诱变方法产生一系列点突变,经过PCR扩增放大和变性复性过程产生异源双链DNA分子,再通过特异性酶切和双色电泳分析识别异源双链中错配碱基,从而检测出突变发生的准确位置。由于具有高通量、大规模、高灵敏度和自动化等特点,能够适应植物功能基因组学研究的要求,TILLING技术已经和即将在功能基因组领域发挥越来越重要的作用。TILLING技术应用于已测序完成的拟南芥和水稻中的突变位点检测并取得了巨大成功;TILLING技术应用于农作物的品种改良,可以帮助实现快速、定向改良作物的品种,同时由于TILLING采用的化学诱变技术与传统诱变育种并无二致,因此在作物改良中采用TILLING技术不存在外源基因转入引发的转基因作物（GMO）争论;由TILLING技术发展来的EcoTILLING技术,具有通量高、成本低、定位准确等优点,可以很好地进行多态性检测和研究基因的功能,已成为开展物种DNA多态性检测和不同物种演替进化研究的有力工具。本文简要介绍了TILLING的原理及操作步骤,讨论了TILLING技术的特点和优点及TILLING技术的应用前景。     

多肽配体技术在植物功能基因组学中的应用前景

人工智能配体或适配体(Aptamer)技术是近年来兴起的一项特异性极强的基因干扰技术。通过人工合成特异的智能配体结合靶基因的蛋白产物, 达到特异干扰靶基因的生物学功能, 这是人工智能配体技术的基本设想。文章综述了多肽配体(Peptide aptamer)技术在基因功能验证中的主要进展, 着重阐明它在植物基因功能验证和作物抗病毒育种中的应用前景, 并提出克服该技术主要风险对策。     

小麦的比较基因组学和功能基因组学

小麦是异源多倍体植物,具有大的染色体组,并且基因组中重复序列所占比例较高,这些特征限制了小麦基因组研究的进展。比较基因组学方法为运用模式植物进行小麦基因组学研究提供了一个操作平台。功能基因组学的研究集中于基因组中转录表达的部分,基因功能的确定是功能基因组学研究的主要内容。对比较基因组学在小麦基因组研究中的应用和小麦功能基因组学的研究内容和方法进行了综述。     

Application of TILLING in Plant Improvement

TILLING (Targeting induced local lesions in genomes) is a general reverse-genetic strategy that is used to locate an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and cost-effective detection of induced point mutations in populations of chemically mutagenized individuals. The technique can be applied not only to model organisms but also to economically important organisms in plants. Owing to its full of advantages such as simple procedure, high sensitivity, and high efficiency, TILLING provides a powerful approach for gene discovery, DNA polymorphism assessment, and plant improvement. Coupled with other genomic resources, TILLING and EcoTILLING can be used immediately as a haplotyping tool in plant breeding for identifying allelic variation in genes exhibiting expression correlating with phenotypes and establishing an allelic series at genetic loci for the traits of interest in germplasm or induced mutants.     

肠道病毒71型的功能基因组学研究进展

肠道病毒71型（enterovirus type 71,EV71）感染通常引起婴幼儿手足口病（hand,foot and mouth disease,HFMD）,但少数可引起无菌性脑膜炎（asepic meningitis）、脑炎（encephalitis）和类脊髓灰质炎的麻痹性疾病（poliomyelitis-like paralysis）等严重的神经系统疾病。功能基因组学研究对于探讨EV71的感染及复制过程、药物及疫苗的研制具有重大意义。该文就EV71的基因组结构及其功能的研究进展作简要的概述。     

功能基因组学的研究方法

基因组学的研究已从结构基因组学转向功能基因组学,功能基因组学时代对于基因功能的研究也由单一基因转向大规模,批量分析,本综述了功能基因组学的研究内容与方法,主要包括：差异显示反转录PCR,基因表达序列分析（SAGE）,微点阵,遗传足迹法,反求遗传学,蛋白质组学和生物信息学等新方法。     

SPR技术与功能基因组和蛋白质组研究

表面等离子体共振(surface plasmon resonance,SPR)依据光学—介质相互作用原理建立,属于实时和非标记的测试方法。SPR方法在研究分子间相互作用方面具有其独特的优势,其非标记和实时检测以及可以进行动力学分析的特点,给研究生物大分子的相互作用提供了诱人的解决方案。近来,随着SPR成像技术和SPR芯片制备技术的进展,将为功能基因组学和蛋白质组学研究提供重要的新的技术平台。     

A transgenic perspective on plant functional genomics

Transgenic crops are very much in the news due to the increasing public debate on their acceptance. In the scientific community though, transgenic plants are proving to be powerful tools to study various aspects of plant sciences. The emerging scientific revolution sparked by genomics based technologies is producing enormous amounts of DNA sequence information that, together with plant transformation methodology, is opening up new experimental opportunities for functional genomics analysis. An overview is provided here on the use of transgenic technology for the functional analysis of plant genes in model plants and a link made to their utilization in transgenic crops. In transgenic plants, insertional mutagenesis using heterologous maize transposons or Agrobacterium mediated T-DNA insertions, have been valuable tools for the identification and isolation of genes that display a mutant phenotype. To discover functions of genes that do not display phenotypes when mutated, insertion sequences have been engineered to monitor or change the expression pattern of adjacent genes. These gene detector insertions can detect adjacent promoters, enhancers or gene exons and precisely reflect the expression pattern of the tagged gene. Activation tag insertions can mis-express the adjacent gene and confer dominant phenotypes that help bridge the phenotype gap. Employment of various forms of gene silencing technology broadens the scope of recovering knockout phenotypes for genes with redundant function. All these transgenic strategies describing gene-phenotype relationships can be addressed by high throughput reverse genetics methods that will help provide functions to the genes discovered by genome sequencing. The gene functions discovered by insertional mutagenesis and silencing strategies along with expression pattern analysis will provide an integrated functional genomics perspective and offer unique applications in transgenic crops. This revised version was published online in August 2006 with corrections to the Cover Date.     

Functional genomics as a tool in virus research

Genomics is the study of an organism’s entire genome. It started out as a great scientific endeavor in the 1990s which aimed to sequence the complete genomes of certain biological species. However viruses are not new to this field as complete viral genomes have routinely been sequenced since the past thirty years. The ‘genomic era’ has been said to have revolutionized biology. This knowledge of full genomes has created the field of functional genomics in today’s post-genomic era, which, is in most part concerned with the studies on the expression of the organism’s genome under different conditions. This article is an attempt to introduce its readers to the application of functional genomics to address and answer several complex biological issues in virus research.     

嗜热四膜虫——具有发展潜力的功能基因组学研究模型

在真核生物的分子生物学和遗传学研究方面,纤毛类原生动物嗜热四膜虫(Tetrahymenathermophila)已经被证明是一种有价值的生物学模型。通过对它的研究,人们发现并且掌握了核酶的分子机制、RNA的自我拼接、端粒的结构和功能、DNA序列重组等机理。这种原生动物的基因组功能分别由两个细胞核执行,即二倍体的小核与生殖过程有关,而多倍体的大核决定细胞的基因转录,并为转化基因的表达提供了强有力的手段。     

基因组信息的整合与植物功能基因组学研究的策略

近年来,随着许多植物基因组测序和可利用序列的增加,相继建立了一些基于靶基因诱变的“反向”遗传学研究策略,如T—DNA诱变、基因敲除、基因沉默和超表达分析等。同时,DNA微阵列和基因芯片技术的发展使得快速、定量检测植物发育不同时期和不同组织器官的基因转录时空变化成为现实。作图技术的改进和来自不同物种基因组信息的整合也正在加速图谱克隆程序的简化和发展。因此,随着生物基因组测序工作日益增多,整合不同类群植物基因组的信息和资源,在植物功能基因组学研究中的重要性日趋显著。     

Characterization of Arabidopsis thaliana mismatch specific endonucleases: application to mutation discovery by TILLING in pea

Scanning DNA sequences for mutations and polymorphisms has become one of the most challenging, often expensive and time-consuming obstacles in many molecular genetic applications, including reverse genetic and clinical diagnostic applications. Enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA at the mismatch sites. These methods are often limited by the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in a pool of DNA and their costs. Here, we present detailed biochemical analysis of five Arabidopsis putative mismatch-specific endonucleases. One of them, ENDO1, is presented as the first endonuclease that recognizes and cleaves all types of mismatches with high efficiency. We report on a very simple protocol for the expression and purification of ENDO1. The ENDO1 system could be exploited in a wide range of mutation diagnostic tools. In particular, we report the use of ENDO1 for discovery of point mutations in the gibberellin 3beta-hydrolase gene of Pisum sativum. Twenty-one independent mutants were isolated, five of these were characterized and two new mutations affecting internodes length were identified. To further evaluate the quality of the mutant population we screened for mutations in four other genes and identified 5-21 new alleles per target. Based on the frequency of the obtained alleles we concluded that the pea population described here would be suitable for use in a large reverse-genetics project.     

RNAi技术及在植物功能基因组研究中的应用

RNAi,即RNA干扰,是通过外源或内源性的双链RNA在细胞内诱导同源序列的基因表达受抑的现象,自RNAi现象在上世纪90年代中期被发现以来,它就被利用到基因组功能的分析研究之中,成为分子生物学研究的热点。本文简要阐述了RNAi的作用机制,并通过将RNAi方法与其他功能基因组研究方法的对比,阐述了RNAi作为高通量植物功能基因组研究方法的优点,同时综述了利用RNAi技术在植物功能基因组研究中的应用进展。     

Integration of structural and functional genomics

This paper introduces a special issue of Animal Genetics , which is devoted to the recent symposium held at Iowa State University entitled 'Integration of Structural and Functional Genomics'. We describe issues and needs that confront the animal genomics community, and describe how this symposium was structured to address these issues by improving communication and collaboration across species and disciplines. The session topics and oral presentations are briefly described for all invited speakers.