植物转座子及其在功能基因组学中的应用

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,且因其 独特的性质已成为发现新基因和基因功能分析的有效工具。本文综述了植物转座子及其作为基因分离和克隆工具的研究进展,并讨论其在植物基因功能研究方面的应用。 Abstract:Transposons have been widely used for gene isolation and gene cloning as insertional mutagens or molecular tagging.Furthermore,due to special characteristics of transposons,transposons techniques will be a powerful tool for new gene discovery and gene functional analysis.This paper reviewed the developments of plant transposons in gene isolating and cloning,as well as its use in studying gene function in plant.     

植物的功能基因组学研究进展 Progress in Functional Genomics in Plants

基因组研究计划包括以全基因组测序为目标的结构基因组学和以基因功能鉴定为目标的功能基因组学两方面的内容。目前基因功能鉴定的方法主要有：基因表达的系统分析(SAGE)、cDNA微阵列、DNA(基因)芯片、蛋白组技术以及基于转座子标签和T_DNA标签的反求遗传学技术等。本文对上述各种技术的优缺点以及它们在植物基因功能鉴定中的应用进行了综述。 Abstract: The genome projects comprise the structural genomics focusing on determining the complete sequences of the genome and the functional genomics focusing on elucidating the biological function of genes.The rapidly evolving tools for functional genomics research include Serial Analysis of Gene Expression (SAGE),cDNA microarray,DNA (or gene) chips,proteome project and the reverse genetics technique based on the well-established transposon tagging and T?DNA tagging systems.In this paper,the advantages and disadvantages of such techniques and application of these techniques in plant functional genomics research are reviewed and future prospective are also presented.     

植物的功能基因组学研究进展

基因组研究计划包括以全基因组测序为目标的结构基因组学和以基因功能鉴定为目标的功能基因组学两方面的内容。目前基因功能鉴定的方法主要有：基因表达的系统分析(SAGE)、cDNA微阵列、DNA(基因)芯片、蛋白组技术以及基于转座子标签和T_DNA标签的反求遗传学技术等。本文对上述各种技术的优缺点以及它们在植物基因功能鉴定中的应用进行了综述。 Abstract: The genome projects comprise the structural genomics focusing on determining the complete sequences of the genome and the functional genomics focusing on elucidating the biological function of genes.The rapidly evolving tools for functional genomics research include Serial Analysis of Gene Expression (SAGE),cDNA microarray,DNA (or gene) chips,proteome project and the reverse genetics technique based on the well-established transposon tagging and T?DNA tagging systems.In this paper,the advantages and disadvantages of such techniques and application of these techniques in plant functional genomics research are reviewed and future prospective are also presented.     

Widely predicting specific protein functions based on protein-protein interaction data and gene expression profile

GESTs (gene expression similarity and taxonomy similarity), a gene functional prediction approach previously proposed by us, is based on gene expression similarity and concept similarity of functional classes defined in Gene Ontology (GO). In this paper, we extend this method to protein-protein interac-tion data by introducing several methods to filter the neighbors in protein interaction networks for a protein of unknown function(s). Unlike other conventional methods, the proposed approach automati-cally selects the most appropriate functional classes as specific as possible during the learning proc-ess, and calls on genes annotated to nearby classes to support the predictions to some small-sized specific classes in GO. Based on the yeast protein-protein interaction information from MIPS and a dataset of gene expression profiles, we assess the performances of our approach for predicting protein functions to “biology process” by three measures particularly designed for functional classes organ-ized in GO. Results show that our method is powerful for widely predicting gene functions with very specific functional terms. Based on the GO database published in December 2004, we predict some proteins whose functions were unknown at that time, and some of the predictions have been confirmed by the new SGD annotation data published in April, 2006.     

Graft‐accelerated virus‐induced gene silencing facilitates functional genomics in rose flowers

Rose has emerged as a model ornamental plant for studies of flower development, senescence, and morphology, as well as the metabolism of floral fragrances and colors.Virus-induced gene silencing(VIGS) has long been used in functional genomics studies of rose by vacuum infiltration of cuttings or seedlings with an Agrobacterium suspension carrying TRV-derived vectors. However, VIGS in rose flowers remains a challenge because of its low efficiency and long time to establish silencing. Here we present a novel and rapid VIGS method that can be used to analyze gene function in rose,called ‘graft-accelerated VIGS', where axil ary sprouts are cut from the rose plant and vacuum infiltrated with Agrobacterium. The inoculated scions are then grafted back onto the plants to flower and silencing phenotypes can be observed within 5 weeks, post-infiltration. Using this new method, we successfully silenced expression of the RhDFR_1, RhA G, and RhNUDX_1 in rose flowers, and affected their color, petal number, as well as fragrance, respectively. This grafting method will facilitate high-throughput functional analysis of genes in rose flowers. Importantly, it may also be applied to other woody species that are not currently amenable to VIGS by conventional leaf or plantlet/seedling infiltration methods.     

An Efficient Rice Mutagenesis System Based on Suspension-Cultured Cells

Plant mutants are important bio-resources for crop breeding and gene functional studies. Conventional methods for generating mutant libraries by mutagenesis of seeds with physical or chemical agents are of low efficiency. Here, we developed a highly-efficient ethyl methanesulfonate (EMS) mutagenesis system based on suspension-cultured cells, with rice (Oryza sativa L.) as an example. We show that treatment of suspension-cultured tiny cell clusters with 0.4% EMS for 18-22h followed by differentiation and regeneration produced as high as 29.4% independent mutant lines with visible phenotypic variations, including a number of important agronomic traits such as grain size, panicle size, grain or panicle shape, tiller number and angle, heading date, male sterility, and disease sensitivity. No mosaic mutant was observed in the mutant lines tested. In this mutant library, we obtained a mutant with an abnormally elongated uppermost internode. Sequencing and functional analysis revealed that this is a new allelic mutant of eui (elongated uppermost internode) caused by two point mutations in the first exon of the EUI gene, representing a successful example of this mutagenesis system.     

Construction and Application of Efficient Ac-Ds Transposon Tagging Vectors in Rice

Transposons are effective mutagens alternative to T-DNA for the generation of insertional mutants in many plant species including those whose transformation is inefficient. The current strategies of transposon tagging are usually slow and labor-intensive and yield low frequency of tagged lines. We have constructed a series of transposon tagging vectors based on three approaches： （i） AcTPase controlled by glucocorticoid binding domain/VP16 acidic activation domain/Gal4 DNA-binding domain （GVG） chemical-inducible expression system; （ii） deletion of AcTPase via Cre-lox site-specific recombination that was initially triggered by Ds excision; and （iii） suppression of early transposition events in transformed rice callus through a dual-functional hygromycin resistance gene in a novel Ds element （HPT-Ds）, We tested these vectors in transgenic rice and characterized the transposition events. Our results showed that these vectors are useful resources for functional genomics of rice and other crop plants. The vectors are freely available for the community,     

A network partition algorithm for mining gene functional modules of colon cancer from DNA microarray data

Computational analysis is essential for transforming the masses of microarray data into a mechanistic understanding of cancer. Here we present a method for finding gene functional modules of cancer from microarray data and have applied it to colon cancer. First, a colon cancer gene network and a normal colon tissue gene network were constructed using correlations between the genes. Then the modules that tended to have a homogeneous functional composition were identified by splitting up the network. Analysis of both networks revealed that they are scale-free. Comparison of the gene functional modules for colon cancer and normal tissues showed that the modules＇ functions changed with their structures.     

Identification and isolation of Mu-flanking fragments from maize

Transposable elements have been utilized as mutagens to create mutant libraries for functional genomics.Isolation of genomic seg-ments flanking the insertion Mutator (Mu) is a key step in insertion mutagenesis studies.Herein,we adopted a modified AFLP method to identify and isolate Mu-flanking fragments from maize.The method consists of the following steps: 1) double-digestion of genomic DNA with Bgl ⅡMsp Ⅰ and ligation of digested fragments to the Bgl Ⅱ- and Msp Ⅰ-adaptors; 2) enrichment of a subset of Bgl Ⅱ/Msp Ⅰ fragments followed by selective amplification of the Mu-flanking fragments; 3) simultaneous display of AFLP bands derived from the flanking re-gions for both insert and native Mu transposons; 4) identification and isolation of AFLP bands resulting from Mu insertions by comparing the banding profiles between Mu-induced mutants and their parental lines; and 5) confirmation of flanking fragments related to these Mu insertions.Using this approach,we have isolated flanking fragment(s) resulting from Mu insertion for every Mu-indueed mutant,and one such fragment,M196-FF,is found to contain a partial sequence of the DNA topoisomerase Ⅰ gene Topl.Moreover,the modified AFLP method including all restriction enzymes,adaptors and primers has been optimized in this study.The modified AFLP method has been proved to be simple and efficient in the isolation of Mu-flanking fragments and will find its usefulness in the functional genomics of maize.     

激发标签技术在植物功能基因组研究中的应用

获得突变体是研究植物功能基因组的有效方法。与传统的T-DNA插入功能缺失突变相比, 建立在功能获得突变基础之上的激发标签技术具有独特的优势, 主要表现为可以获得功能冗余基因的显性突变体并方便地克隆相关基因。文章对激发标签技术的原理, 及其在拟南芥、水稻等植物功能基因组研究中的进展进行了综述, 并介绍了激发标签技术在植物抗逆、抗病和生长发育机理研究方面的新进展。文章最后探讨了激发标签技术的发展前景。     

激活标签法构建拟南芥突变体库及其表型分析

以拟南芥(ArabMopsis thaliana)野生生态型(Columbia)植株为实验材料,以含有激活标记双元质粒pCB260的农杆菌进行转化,并以抗除草剂Basta为筛选标记,构建了拟南芥激活标签突变体库,所用pCB260双元质粒含有两个Ds位点、一个GFP标记基因与一个抗basta标记基因,可以方便高效地筛选转基因植物.目前经初步筛选获得了约10 000个独立转化株系(T1代),其中约50个株系具有明显的表型变化,包括花期改变、株型变异、叶形特异、育性降低、花发育异常、种子颜色变浅等.运用TAIL-PCR技术,成功获得了其中10个表型特异株系的T-DNA侧翼序列,分别分布于拟南芥基因组的5条染色体上.     

Constructing and Phenotypic Analyzing an Activation Tagging Arabidopsis Mutant Pool

Activation tagging method is an effective tool of obtaining gain of function mutant and investigating the gene function, which plays an important role in plant functional genomics study. In this paper, we used Arabidopsis Columbia wild type as material to construct an activation tagging mutant pool by Agrobacterium tumefaciens mediated transformation, the binary vector pCB260 contained two Ds elements, one GFP report gene and one basta resistance selection genes, which show more convenient and efficient to screen the transgenic plant. Until now, over ten thousand transformed plants were generated. Among them, about 50 dominant mutants with obvious phenotypes were isolated, including early or late flowering time, unmoral leaf shape and flower, sterility and thin seed capsule color. T DNA flanking sequences of ten special mutants were validated by TAIL PCR and sequencing, whose T DNA insertion fragments distributed in all five chromosomes of Arabidopsis genome, respectively.     

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。     

Transposon display for active DNA transposons in rice

Transposon display (TD) is a powerful technique to identify the integration site of transposons in gene tagging as a functional genomic tool for elucidating gene function. Although active endogenous DNA transposons have been used extensively for gene tagging in maize, only two active endogenous DNA transposons in rice have been identified, the 0.43-kb element mPing of the MITE family and the 0.6-kb nDart element of the hAT family. The nDart transposition was shown to be induced by crossing with a line containing its autonomous element aDart and stabilized by segregating aDart under natural growth conditions, while mPing-related elements were shown to transpose in cultured cells, plants regenerated from an anther culture, and gamma-ray-irradiated plants. No somaclonal variation should occur in nDart-promoted gene tagging because no tissue culture was involved in nDart activation. As an initial step to develop an effective tagging system using nDart in rice, we tried to visualize GC-rich nDart-related elements comprising 18 nDart-related sequences of 0.6-kb and 63 nDart-related elements longer than 2 kb in Nipponbare by TD. Comparing the observed bands in TD with the anticipated virtual bands of the nDart-related elements based upon the available rice genome sequence, we have improved our TD protocol by optimizing the PCR amplification conditions and are able to visualize approximately 87% of the anticipated bands produced from the nDart-related elements. To compare the visualization efficiency of these nDart-related elements with that of 50 mPing elements and a unique Ping sequence in Nipponbare, we also tried to visualize the mPing-related elements; all mPing-related elements are easily visualized. Based on these results, we discuss the parameters affecting the visualization efficiencies of these rice DNA transposons. We also discuss the utilization of nDart elements in gene tagging for functional genomics in rice.     

Genetic resources offer efficient tools for rice functional genomics research

Rice is an important crop and major model plant for monocot functional genomics studies. With the establishment of various genetic resources for rice genomics, the next challenge is to systematically assign functions to predicted genes in the rice genome. Compared with the robustness of genome sequencing and bioinformatics techniques, progress in understanding the function of rice genes has lagged, hampering the utilization of rice genes for cereal crop improvement. The use of transfer DNA (T‐DNA) insertional mutagenesis offers the advantage of uniform distribution throughout the rice genome, but preferentially in gene‐rich regions, resulting in direct gene knockout or activation of genes within 20–30 kb up‐ and downstream of the T‐DNA insertion site and high gene tagging efficiency. Here, we summarize the recent progress in functional genomics using the T‐DNA‐tagged rice mutant population. We also discuss important features of T‐DNA activation‐ and knockout‐tagging and promoter‐trapping of the rice genome in relation to mutant and candidate gene characterizations and how to more efficiently utilize rice mutant populations and datasets for high‐throughput functional genomics and phenomics studies by forward and reverse genetics approaches. These studies may facilitate the translation of rice functional genomics research to improvements of rice and other cereal crops.     

Screening for mutations affecting sexual reproduction after activation tagging inArabidopsis thaliana

In this work, a seed-set-based screening was performed on 70 lines of Arabidopsis thaliana after activation tagging mutagenesis to identify mutations in reproductive mechanisms. Five mutants showed significantly lower seed set than the wild type and confirmed the phenotype in the progeny. This phenotype was linked with the marker gene bar carried by T-DNA conferring glufosinate resistance. Genetic analysis revealed that the mutation inheritance was sporophytic in 3 mutants and gametophytic in 2 mutants. In addition, 2 mutants had an extra T-DNA copy. Thus activation tagging can be an effective strategy to identify new mutations affecting sporogenesis or gametogenesis.