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。     

Searching for tagged male-sterile mutants of arabidopsis

Although many male-sterile mutants have been identified inArbidopsis thaliana, few of the corresponding genes have been cloned. In order to facilitate cloning of a male sterility gene, 23 of Feldmann's T-DNA-generated, reduced-fertility lines were screened to identify a tagged male-sterile mutation. Malesterile mutants were identified, as well as mutants that were both male and female sterile. Segregation of the kanamycin marker gene in the progeny of 15 of these lines was studied. Forty percent had functional T-DNAs (encoding resistance to kanamycin) inserted at a single locus, the remainder segregating for two or more functional T-DNA inserts. Linkage between T-DNA inserts and mutant phenotype was tested for six lines. In three of these lines, mutations were not linked to a T-DNA insert. In three lines, the mutation segregated with a T-DNA insert.     

T-DNA as a gene tag

The characteristics of Agrobacterium-mediated plant transformation are such that the T-DNA can be used as an insertional mutagen and, by extension, a gene tag. An increasing number of mutants have been obtained as a result of T-DNA insertion and the versatility of this experimental system can be exploited further to tag sequences of DNA which control gene expression. While the isolation of specific mutations by T-DNA tagging in the past has been fortuitous, T-DNA-based vectors have the potential to generate transgenic plants with specific, selectable mutations.     

T-DNA标签在植物基因克隆和功能分析中的应用

在植物功能基因组学的研究中,插入突变已成为迅速识别和研究标签基因的一个有效遗传工具.本文介绍了T-DNA标签的概念及应用前提,详细论述了T-DNA标签在大规模植物基因功能分析中的应用以及使用启动子和增强子诱捕技术分离时空特异性启动子和表达基因,另外还分析了利用其特殊形式激活标签进行基因克隆和功能分析的优越性,并展望了T-DNA标签的应用前景.     

拟南芥光形态突变体的筛选与基因鉴定

以哥伦比亚(Columbia)野生型拟南芥(Arabidopsis thaliana)为实验材料,用含有激活标记双元质粒pCB260的农杆菌浸花进行转化,构建拟南芥T-DNA插入突变体库.通过突变体的筛选和表型分析,获得了两株光形态突变体,子叶下胚轴伸长的光抑制效应减弱.通过TAIL-PCR(thermal asymmetric interlaced-PCR)技术,成功扩增出突变植株T-DNA插入位点侧翼序列,经NCBI序列比对,T-DNA分别插在CRY1第一和第三外显子部位.突变体的表型分析及PCR鉴定结果表明,T-DNA插入CRY1并影响到突变植株的光形态建成.     

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.     

Next-generation sequencing as a tool to quickly identify causative EMS-generated mutations

The advent of next generation sequencing has influenced every aspect of biological research. Many labs are now using whole genome sequencing in Arabidopsis thaliana as a means to quickly identify EMS-generated mutations present in isolated mutants. Following identification of these mutations, examination of T-DNA insertional alleles defective in candidate genes or complementation of the mutant phenotype with a wild type copy of candidate genes can be used to verify which mutation is causative for the phenotype of interest. Here, we discuss the benefits and pitfalls of using this method to identify mutations underlying phenotypes.     

Activation tagging,a novel tool to dissect the functions of a gene family

In a screen for morphological mutants from the T1 generation of approximately 50 000 activation-tagging lines, we isolated four dominant mutants that showed hyponastic leaves, downward-pointing flowers and decreased apical dominance. We designated them isoginchaku (iso). The iso-1D and iso-2D are allelic mutants caused by activation of the AS2 gene. The T-DNAs were inserted in the 3' downstream region of AS2. Iso-3D and iso-4D are the other allelic mutants caused by activation of the ASL1/LBD36 gene. These two genes belong to the AS2 family that is composed of 42 genes in Arabidopsis. The only recessive mutation isolated from this gene family was of AS2, which resulted in a leaf morphology mutant. Applying reverse genetics using a database of activation-tagged T-DNA flanking sequences, we found a dominant mutant that we designated peacock1-D (pck1-D) in which the ASL5/LBD12 gene was activated by a T-DNA. The pck1-D mutants have lost apical dominance, have epinastic leaves and are sterile. These results strongly suggest that activation tagging is a powerful mutant-mining tool especially for genes that make up a gene family.     

Cloning of an Arabidopsis patatin-like gene, STURDY, by activation T-DNA tagging

Activation T-DNA tagging can generate dominant gain-of-function mutants by overexpression of a particular endogenous gene. We identified an activation-tagged mutant, sturdy, exhibiting a stiff inflorescence stem, thicker leaves, shorter siliques, larger seeds, round-shaped flowers, and delayed growth. It is most important that unlike its wild-type counterpart, this mutant is less prone to lodging. Cloning of STURDY revealed that in sturdy, there is an open reading frame containing a single intron encoding a patatin-like homolog. The T-DNA is inserted into the 3' region of the second exon. The mutant phenotype was shown to be the result of overexpression of STURDY by mRNA analysis and transgenic studies. Preliminary histological studies have revealed an increase in cell number in the inflorescence stem of mutant plants; however, additional studies are needed to better understand the overexpression phenotype.     

Activation-Tagged Tobacco Mutants that are Tolerant to Antimicrotubular Herbicides are Cross-Resistant to Chilling Stress

T-DNA activation tagging was used to generate tobacco mutants with increased tolerance to antimicrotubular herbicides and chilling stress. After transformation, protoplast-derived calli were screened for tolerance to treatments that affect microtubule assembly. In one screen mutants with tolerance to aryl carbamates (a blocker of microtubule assembly) were selected, the second screen was targeted to chilling-tolerant mutants that could survive for several months at 3°C, a third screen combined both factors. The resistance of these mutants to aryl carbamates or chilling was accompanied by resistance of microtubules to these factors. The carbamate tolerant mutants were cross-resistant to chilling stress. This was mirrored by an adaptive reorganization of microtubules and a reduction of microtubule dynamics in response to chilling. The analysis of these mutants suggests (1) that microtubule dynamics limit the tolerance to chilling and EPC, and (2) that the cold sensitivity of microtubules limits chilling tolerance in tobacco.     

水稻T-DNA插入突变体库的筛选及遗传分析

T-DNA标签技术是分离和研究植物功能基因的有效方法,寻找T-DNA插入表型突变体是进一步开展研究的关键所在。文章对以ZH11、ZH15为受体亲本构建的4416份T,代标签系进行了表型鉴定,发现存在拟纯合突变和系内分离突变两种类型,突变表型涉及株高、生育期、叶形、叶色、分蘖力、植株松紧度、穗颈节、穗形、颖花、粒形、类病变、雄性不育、生长极性等14类性状。其中,株高、生育期、叶色、雄性不育有着相对较高的突变频率（超过1％）,株高和叶色的突变频率在品种及年度间表现稳定,而生育期、雄性不育波动较大,表明这类性状的表型易受到环境的影响。通过T1、T2连续世代的共分离分析,筛选出3个与穗部或颖花发育相关的T-DNA插入突变体,为分离相关功能基因奠定基础。随机选择42份有表型突变的标签系,通过质粒拯救和TAIL-PCR的方法分离其侧翼序列,从39个标签系中获得40条序列,其中25条为载体序列,14条与水稻基因组有很好的同源性,BlastN分析结果表明T-DNA有优先整合进植物功能基因内部的特性。     

Screening for and Genetic Analysis on T-DNA-inserted Mutant Pool in Rice

T-DNA tagging technique has provided a powerful strategy for identifying new functional genes in plants, and the key for success is the discovery of T-DNA-inserted mutants with changed phenotype. In this study, we screened 4 416 rice T₁ tagged lines generated by enhancer trap system integrated with GLL4/VP16-UAS elements from two transformed parents, ZH11 and ZH15. We found many lines showed obvious morphological mutations, including two types—fake-homozygous mutation and separating mutation. The mutation phenotype was related to 14 kinds of trait such as plant height, heading date, leaf shape, leaf color, tiller number, panicle shape, spikelet number, grain shape, disease-like mutation, male sterility, awn, and so on. Among them, plant height, heading date, leaf color and male sterility had a comparatively high mutation frequency (over 1%). The mutation frequency of plant height and leaf color had no significant change between different years or transformed parents, but the frequency of heading date and male sterility varied greatly, suggesting that environment had a great effect on the expression of latter two traits. By conducting continuously co-segregating analyses in T₁ and T₂ generation, we identified 3 T-DNA-inserted mutants with malformed panicle or spikelets, which would provide a base for cloning correlative functional genes. At the same time, we selected randomly 42 lines with mutation phenotype and obtained 40 flanking sequences from 39 tagged lines by plasmid rescue or TAIL-PCR, of which, 26 were vector backbone sequence, 14 had good identity to rice genome sequence. The BlastN result showed the T-DNA preferentially integrated into protein-coding region in plants.