拟南芥激活标记突变体库的构建及突变体基因的克隆

激活标记(activation tagging)技术是以功能获得突变体为研究对象,在植物功能基因组学的研究中具有重要的作用.文章以双子叶模式植物拟南芥(Arabudidopsis thaliana)野生生态型植株为实验材料,以含有激活标记质粒pSKI015的农杆菌直接喷雾进行转化,并以抗除草剂Basta为筛选标记,构建了拟南芥的激活标记突变体库.结果共得到约20 000个独立转化株系(T1代),其中38个株系有明显的表型变化,约占转化植株总数的千分之二.基因组DNA Southern杂交结果表明,大多数转化植株为多拷贝T-DNA插入.通过质粒拯救(plasmid rescue)和TAIL-PCR(Thermal asymmetric interlaced-PCR)可获得T-DNA插入的基因组旁邻序列,为克隆突变体的基因奠定基础.     

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.     

利用双T-DNA载体系统培育无选择标记转基因烟草

建立了一种利用双T-DNA载体培育无选择标记转基因植物的方法.通过体外重组构建了双T-DNA双元载体pDLBRBbarm.载体中,选择标记nptⅡ基因和另一代表外源基因的bar基因分别位于2个独立的T-DNA.利用农杆菌介导转化烟草(Nicotiana tabacum L.),在获得的转化植株中,同时整合有nptⅡ基因和bar基因的频率为59.2%.对4个同时整合有nptⅡ和bar基因植株自交获得的T1代株系进行检测分析,发现在3个T1代株系2个T-DNA可以发生分离,其中约19.5%的转基因T1代植株中只存在bar基因而不带选择标记nptⅡ.这一结果说明双T-DNA载体系统能有效地用于培育无选择标记的转基因植物.研究还利用位于2个不同载体上的nptⅡ基因与 bar基因通过农杆菌介导共转化烟草,获得共转化植株的频率为20.0%～47.4%,低于使用双T-DNA转化的共转化频率.     

小麦TaCO9基因的克隆及功能分析

为了明确TaCO9-1A基因在小麦生长发育中的具体调控机理,该研究以同源克隆的方法成功获得了大麦(Hordeum vulgare)光周期基因HvCO9在小麦(Triticum aestivum)中的直系同源基因TaCO9,并对其进行生物信息学分析、亚细胞定位、转录激活以及表达模式分析;利用农杆菌侵染法转化拟南芥,并对过表达株系进行表型分析。结果表明:(1)TaCO9包含2个外显子和1个内含子,CDS区全长876 bp,编码291个氨基酸,蛋白序列含有特有的CCT结构域,且在不同物种间高度保守。(2)生物信息学分析表明,TaCO9基因编码的蛋白质分子量约为30.7 kD,等电点为6.24;TaCO9的启动子区含有光响应、激素应答和胁迫应答等多种顺式作用元件。(3)亚细胞定位和转录活性分析表明,TaCO9主要定位于细胞核中,且具有转录激活活性。(4)qRT-PCR结果表明,TaCO9基因在各个组织中均有表达,叶片中的表达量最高;在光照14 h条件下TaCO9的表达量显著高于光照10 h和12 h条件下的表达量,且TaCO9在大粒小麦‘西农817’子房与籽粒中的表达量显著高于‘中国春’。(5)经潮霉素筛选成功获得3个转基因拟南芥株系;进一步功能鉴定结果表明,过表达转TaCO9基因拟南芥植株的开花期迟于野生型(对照)3~4 d,但角果和籽粒较野生型大。     

病程相关基因启动子片段与GUS的融合基因在拟南芥中的表达

PR1是拟南芥(Arabidopsisis thaliana L.)系统获得抗性的一个标志基因.利用PCR技术,从拟南芥中扩增并克隆了PR1基因的启动子片段.将该启动子片段与GUS报告基因拼接,构建成含有PR1-GUS融合基因的重组表达质粒.经根癌农杆菌介导转化,得到了转基因的拟南芥植株.用已知的系统获得抗性激活剂处理转基因植物,检测到GUS活性.因此,这一转基因体系可以作为一种简便、灵敏的实验体系以筛选激活植物系统获得抗性的化合物.     

甘蔗花叶病毒E株系外壳蛋白基因原核表达载体的构建与表达

目的:用原核表达的方法获取大量带6个His标记的甘蔗花叶病毒E株系(ScMV-E)外壳蛋白(CP)。方法:用带有BamHⅠ和SalⅠ酶切位点的特异引物,以带有多个基因的重组质粒pNUSCP为模板,扩增出片段长度为942bp的ScMV-E外壳蛋白基因,亚克隆到pMD18-T载体上,转化E.coliDH5α,经双酶切检测获得阳性克隆。BamHⅠ和SalⅠ双酶切阳性克隆质粒,回收目的片段ScMV-E的CP基因。把目的片段插入表达载体pET29a( ),转化E.coliBL21(DE3),测序。结果:阳性质粒pET29a-CP在E.coliBL21(DE3)中得到大量特异表达。SDS-PAGE分析表明,该蛋白的相对分子质量约36000,与预测一致。结论:以上方法可以得到带6个His标记的目的蛋白,有利于纯化并获取高纯度的ScMV-E的外壳蛋白。     

化学诱导激活型拟南芥突变体库的构建及分析

利用化学诱导激活XVE（LexA-VP16-ER）系统构建了一个包含40000余个独立转化株系的拟南芥突变体库,并对其中的18000余个株系进行了初步的遗传学和表型分析鉴定。卡那霉素抗性分离比表明,51．6％的株系为单位点插入株系,T-DNA插入的平均拷贝数为每株系1．38个。部分T1代和T2代植株表现出了可见的形态变异,包括下胚轴长度、根长度、植株大小和颜色、叶子颜色和形态、开花时间、种皮颜色及结实情况等对数个代表性突变株系表型及T—DNA插入位点侧翼序列进行了分析,结果表明突变体的表型是由于T—DNA的插入造成的,而且这些突变体中包括前人发现的AP2和AGAMOUS的等位基因。由于T-DNA标记或相邻的基因可被XVE系统诱导性的激活,或被T-DNA破坏导致功能缺失,该突变体库可以用于大规模筛选鉴定功能缺失性和功能获得性突变体。     

病程相关基因启动子片段与GUS的融合基因在拟南芥中的表达

PR1是拟南芥 (Arabidopsis thaliana L.) 系统获得抗性的一个标志基因。利用PCR技术,从拟南芥中扩增并克隆了PR1基因的启动子片段。将该启动子片段与GUS报告基因拼接,构建成含有PR1-GUS融合基因的重组表达质粒。经根癌农杆菌介导转化,得到了转基因的拟南芥植株。用已知的系统获得抗性激活剂处理转基因植物,检测到GUS活性。因此,这一转基因体系可以作为一种简便、灵敏的实验体系以筛选激活植物系统获得抗性的化合物。     

利用双T-DNA载体系统培育无选择标记转基因烟草

建立了一种利用双T-DNA载体培育无选择标记转基因植物的方法。通过体外重组构建了双T-DNA双元载体pDLBRBbarm。载体中,选择标记nptⅡ基因和另一代表外源基因的bar基因分别位于2个独立的T-DNA。利用农杆菌介导转化烟草(Nicotiana tabacum L.),在获得的转化植株中,同时整合有nptⅡ基因和bar基因的频率为59．2％。对4个同时整合有nptⅡ和bar基因植株自交获得的T1代株系进行检测分析,发现在3个T1代株系2个T-DNA可以发生分离,其中约19．5％的转基因T1代植株中只存在bar基因而不带选择标记nptⅡ。这一结果说明双T-DNA载体系统能有效地用于培育无选择标记的转基因植物。研究还利用位于2个不同载体上的nptⅡ基因与bar基因通过农杆菌介导共转化烟草,获得共转化植株的频率为20．0％～47．4％,低于使用双T-DNA转化的共转化频率。     

利用转基因标记NPTⅡ快速、规模化纯合转基因番茄

利用卡那霉素喷施方法对带有NPTⅡ标记基因和双价外源基因(烟草渗调蛋白基因AP24和菜豆几丁质酶基因Chi)的转基因番茄的3个世代在温室或田间环境进行规模化筛选,成功获得8个单拷贝转基因纯合植株。含有单个拷贝NPTⅡ标记基因的转基因株系后代,对卡那霉素的抗感分离符合3∶1的孟德尔分离比例,T2代中一些株系表现为对卡那霉素全抗,表明这些株系的外源基因已经纯合,这一结果在T3代中进一步得到证实。但对于含有两个拷贝外源基因的转基因株系,外源基因的遗传则比较复杂。同时,结合Km喷施和多重PCR技术对外源基因的异常遗传进行了初步分析。用PCR分析进一步证实了该方法的准确性,该方法是对转基因番茄进行大规模、快速遗传分析的理想方法。     

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.     

Efficient screening of Arabidopsis T-DNA insertion lines using degenerate primers

The sequencing of the Arabidopsis plant genome is providing a fuller understanding of the number and types of plant genes. However, in most cases we do not know which genes are responsible for specific metabolic and signal transduction pathways. Analysis of gene function is also often confounded by the presence of multiple isoforms of the gene of interest. Recent advances in PCR-based reverse genetic techniques have allowed the search for plants carrying T-DNA insertions in any gene of interest. Here we report preliminary screening results from an ordered population of nearly 60,470 independently derived T-DNA lines. Degenerate PCR primers were used on large DNA pools (n = 2,025 T-DNA lines) to screen for more than one gene family member at a time. Methods are presented that facilitated the identification and isolation of isoform-specific mutants in almost all members of the Arabidopsis H(+)-proton ATPase gene family. Multiple mutant alleles were found for several isoforms.     

拟南芥矮小丛生突变体的分离与分子鉴定

顶端优势是指侧生分生组织的生长被主茎或主花序所抑制.最近的研究通过分离和鉴定顶端优势发生改变的突变体开始揭示顶端优势的分子机制.通过T-DNA标签法分离了拟南芥矮小丛生(bushy and dwarf 1, bud1 )突变体.突变体植株的表型包括顶端优势丧失、株型矮小,表明bud1 突变体存在生长素代谢、运输或信号传导的缺陷.一个对生长素特异反应的启动子驱动的报告基因在bud1 中表达模式改变.生长素敏感性和运输能力的测定表明这两个过程在 bud1中均正常.以上结果显示bud1 表型是生长素代谢缺陷的结果.遗传分析表明BUD1 为半显性突变且与一个T-DNA插入共分离,可通过iPCR方法分离.     

提高水稻插入突变体库利用效率的尝试

T-DNA标签法是一种以农杆菌介导的遗传转化为基础来创造插入突变体库, 从而高通量地分离和克隆植物功能基因的方法。但由于种种原因, 水稻插入突变体库的利用效率较低。为了提高水稻插入突变体库的利用效率, 结合水稻一个双拷贝T-DNA插入突变体的发现和鉴定研究, 通过特异PCR检测、侧翼序列与目标性状的共分离分析, 在1个双插入位点均为杂合的植株的后代株系中分拆了2个插入事件, 分离出目标性状存在遗传分离且只带有1个插入事件的后代株系, 为后续的共分离检测和基因克隆研究打下了重要的基础。由此产生了对插入突变体库中的非串联多拷贝插入标签系进行研究的一些思路和方法, 提出来与同行商榷。     

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。     

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.     

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.     

Activation tagging using the En-I maize transposon system in Arabidopsis

A method for the generation of stable activation tag inserts was developed in Arabidopsis using the maize (Zea mays) En-I transposon system. The method employs greenhouse selectable marker genes that are useful to efficiently generate large populations of insertions. A population of about 8,300 independent stable activation tag inserts has been produced. Greenhouse-based screens for mutants in a group of plants containing about 2,900 insertions revealed about 31 dominant mutants, suggesting a dominant mutant frequency of about 1%. From the first batch of about 400 stable insertions screened in the greenhouse, four gain-in-function, dominant activation-tagged, morphological mutants were identified. A novel gain-in-function mutant called thread is described, in which the target gene belongs to the same family as the YUCCA flavin-mono-oxygenase that was identified by T-DNA activation tagging. The high frequency of identified gain-in-function mutants in the population suggests that the En-I system described here is an efficient strategy to saturate plant genomes with activation tag inserts. Because only a small number of primary transformants are required to generate an activation tag population, the En-I system appears to be an attractive alternative to study plant species where the present transformation methods have low efficiencies.