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.     

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.     

拟南芥光周期开花突变体的筛选和基因的鉴定

以双子叶模式植物拟南芥（Arabidopsis thaliana）突变体crylcry2为实验材料,用舍有激活标记质粒DSK1015的农杆菌浸花进行转化,构建了拟南芥T-DNA插入突变体库．通过筛选和观察分析,获得了一些开花时间比crylcry2明显延迟或明显提早的突变体．采用IPCR（inverse PCR）和TAIL-PCR（thermal asymmetric interlaced PCR）等方法,鉴定了这些突变体T-DNA插入位点的基因组旁邻序列,并采用半定量RT-PCR对插入位点两侧基因的mRNA水平进行了分析,初步鉴定了与开花相关的候选基因,为进一步研究其功能,深入研究隐花素调节光周期开花的作用机制奠定了基础．     

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。     

拟南芥与大豆疫霉菌的非寄主互作及一个感病突变体的遗传分析

非寄主抗病性是一种普遍的自然现象, 该文通过建立拟南芥-大豆疫霉菌(Arabidopsis thaliana-Phytophthora sojae)非寄主互作系统, 筛选对大豆疫霉菌感病的拟南芥突变体, 为研究植物对卵菌的非寄主抗病性遗传机制奠定基础。以大豆疫霉菌游动孢子接种拟南芥T-DNA插入突变体离体叶片, 从代表12 000个独立转化株系的40 000株T₃代T-DNA插入拟南芥突变体中获得一系列对大豆疫霉菌感病的突变体。其中突变体581-51感病性状表现稳定, 离体叶片接菌后3天内出现明显的水渍状病斑, 4–5天后产生大量卵孢子和/或孢子囊。细胞学观察发现有典型的吸器形成。Southern杂交和遗传分析结果表明, 581-51突变体含有4个T-DNA插入事件, 其感病性状可能由隐性单基因控制。     

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。     

拟南芥与大豆疫霉菌的非寄主互作及一个感病突变体的遗传分析

非寄主抗病性是一种普遍的自然现象,该文通过建立拟南芥．大豆疫霉菌（Arabidopsis thaliana—Phytophthora sojae）非寄主互作系统,筛选对大豆疫霉菌感病的拟南芥突变体,为研究植物对卵菌的非寄主抗病性遗传机制奠定基础。以大豆疫霉菌游动孢子接种拟南芥T—DNA插入突变体离体叶片,从代表12000个独立转化株系的40000株T3代T。DNA插入拟南芥突变体中获得一系列对大豆疫霉菌感病的突变体。其中突变体581-51感病性状表现稳定,离体叶片接菌后3天内出现明显的水渍状病斑,4—5天后产生大量卵孢子和／或孢子囊。细胞学观察发现有典型的吸器形成。Southern杂交和遗传分析结果表明,581—51突变体含有4个T-DNA插入事件,其感病性状可能由隐性单基因控制。     

国外大刊重要论文简介

拟南芥的全基因组插入突变体分析 ①AlonsoJM ,StepanovaAN ,LeisseTJetal.Science,2 0 0 3 ,3 0 1:653～ 657在拟南芥和水稻基因组测序完成后 ,下一步就是研究基因功能和基因调控。功能缺失突变体可用来分析功能未知基因。Alonso等创建序列标记的农杆菌T_DNA插入拟南芥突变体库中 ,最后获得的 15万个转化植物能够表达卡那霉素抗性并且独立繁殖。平均每株中含有 1.5个T_DNA插入片段 ,这意味着拟南芥突变体库大约包含 2 2 50 0 0个成功的T_DNA插入整合 ,几乎代表了拟南芥的整个基因区域。使用一个高通量插入位点恢复系统 ,其中 880…     

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.     

水稻T-DNA插入突变体库的构建及突变类型的分析

利用农杆菌介导的转化系统转化中花11成熟胚愈伤组织,获得1489个独立转化的T-DNA插入再生株系。PCR和Southern杂交的结果表明,69．8％转化株系被整合了T-DNA,通过Tail-PCR也从转化植株中扩增出T-DNA侧翼序列。同时对1066个T1转化株系的抽穗期、株高、单株穗数的调查结果表明,不同株系中分离出了突变植株。     

Tagging Hansenula polymorpha genes by random integration of linear DNA fragments (RALF)

We have investigated the feasibility of using gene tagging by restriction enzyme-mediated integration (REMI) to isolate mutants in Hansenula polymorpha. A plasmid that cannot replicate in H. polymorpha and contains a dominant zeocin resistance cassette, pREMI-Z, was used as the integrative/mutagenic plasmid. We observed that high transformation efficiency was primarily dependent on the use of linearised pREMI-Z, and that the addition of restriction endonuclease to linearised pREMI-Z prior to transformation increased the transformation frequency only slightly. Integration of linearised pREMI-Z occurred at random in the H. polymorpha genome. Therefore, we termed this method Random integration of Linear DNA Fragments (RALF). To explore the potential of RALF in H. polymorpha, we screened a collection of pREMI-Z transformants for mutants affected in peroxisome biogenesis (pex) or selective peroxisome degradation (pdd). Many previously described PEX genes were obtained from the mutant collection, as well as a number of new genes, including H. polymorpha PEX12 and genes whose function in peroxisome biogenesis is still unclear. These results demonstrate that RALF is a powerful tool for tagging genes in H. polymorpha that should make it possible to carry out genome-wide mutagenesis screens.     

Transformation and mutagenesis of the nematode-trapping fungus Monacrosporium sphaeroides by restriction enzyme-mediated integration (REMI)

In this study, the nematode-trapping fungus, Monacrosporium sphaeroides, was transformed with a plasmid harboring the hygromycin B phosphotransferase gene, via restriction enzyme-mediated integration (REMI). Frequencies of up to 94 transformants microg(-1) per linearized plasmid DNA were obtained by optimizing the PEG concentration, as well as the category and quantity of the added restriction enzyme. 90% of the transformants were determined to be stable for drug resistance when 20 randomly selected transformants were tested. Southern analyses revealed that the transforming DNA was integrated into the M. sphaeroides genome either with or without rearrangement. Five mitotic stable mutant strains were obtained using this approach, all of which had been altered with regard to sporulation capacity and pathogenicity toward nematodes. Southern blot analyses of the five mutants revealed that foreign plasmid DNA had integrated into the genome. Three of the mutants, Tms2316, Tms3583 and Tms1536, exhibited integration at a single location, whereas the remaining two, Tms32 and Tms1913, manifested integration at double or multiple locations. Our results suggest that the transformation of M. sphaeroides via REMI will facilitate insertional mutagenesis, the functional analysis of a variety of genes, and the tagging or cloning of genes of interest.     

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.     

利用反义RNA技术分析春化相关基因VER17在冬小麦花发育过程中的功能

为了研究小麦春化相关基因VER17的功能,应用反义RNA技术,将VER17基因的反义片段构建到载体pBI121上,通过花粉管通道法获取转基因小麦.对T0代转基因植株GUS染色以及PCR等分子鉴定,得到14株含反义VERJ7基因片段的阳性转基因植株.对T0代和T1代的表型观察结果显示,VER17反义转基因植株开花时间延迟,并且穗的顶部和基部小花出现明显的退化.表明春化相关基因VER17在小麦发育过程中可能起到促进植物开花以及穗顶端和基部花发育的作用,减少小花退化,同时对雄蕊的发育也有影响.     

水稻Ds插入纯合体的筛选和鉴定

采用Basta抗性鉴定、潮霉素抗性鉴定和PCR检测相结合的方法筛选和鉴定了水稻Ds插入纯合体。在T1代236个转化株系中,有16个株系的全部植株表现出对Basta的敏感,其余220个株系的植株表现出对Basta的抗性。经过3代的纯合筛选,共鉴定出Ds插入纯合体203个．这些Ds插入纯合体可用于构建Ac／Ds系统和对Ds插入突变体进行筛选和鉴定,为水稻功能基因组学研究提供了材料。     

Pheromone-regulated Genes Required for Yeast Mating Differentiation

Yeast cells mate by an inducible pathway that involves agglutination, mating projection formation, cell fusion, and nuclear fusion. To obtain insight into the mating differentiation of Saccharomyces cerevisiae, we carried out a large-scale transposon tagging screen to identify genes whose expression is regulated by mating pheromone. 91,200 transformants containing random lacZ insertions were screened for β-galactosidase (β-gal) expression in the presence and absence of α factor, and 189 strains containing pheromone-regulated lacZ insertions were identified. Transposon insertion alleles corresponding to 20 genes that are novel or had not previously been known to be pheromone regulated were examined for effects on the mating process. Mutations in four novel genes, FIG1, FIG2, KAR5/ FIG3, and FIG4 were found to cause mating defects. Three of the proteins encoded by these genes, Fig1p, Fig2p, and Fig4p, are dispensible for cell polarization in uniform concentrations of mating pheromone, but are required for normal cell polarization in mating mixtures, conditions that involve cell–cell communication. Fig1p and Fig2p are also important for cell fusion and conjugation bridge shape, respectively. The fourth protein, Kar5p/Fig3p, is required for nuclear fusion. Fig1p and Fig2p are likely to act at the cell surface as Fig1:: β-gal and Fig2::β-gal fusion proteins localize to the periphery of mating cells. Fig4p is a member of a family of eukaryotic proteins that contain a domain homologous to the yeast Sac1p. Our results indicate that a variety of novel genes are expressed specifically during mating differentiation to mediate proper cell morphogenesis, cell fusion, and other steps of the mating process.     

Transformation of rat fibroblast cells with early mutants of polyoma (tsa) and simian virus 40 (tsA30): occurrence of either A or N transformants depends on the multiplicity of infection.

Infection of rat fibroblasts with early mutants of polyoma virus (tsa) or simian virus 40 (tsA30) leads to the establishment of either temperature-independent A transformants or N transformants temperature-sensitive for the expression of the transformed phenotype. The choice between the A- and N-transformed states is not only dependent, as we reported previously (Rassoulzadegan et al., j. Virol., 28:421-426, 1978), on the growth conditions after infection, but is also a function of the multiplicity of infection (MOI); high MOI led to the predominant occurrence of A derivatives, and lower MOI led to that of N transformants.     

Development of an activation tagging system for the basidiomycetous medicinal fungus Antrodia cinnamomea

This study describes the development of an efficient and reliable activation tagging system for the medicinal fungus Antrodia cinnamomea. For successful Agrobacterium tumefaciens-mediated transformation, different parameters were considered. The Agrobacterium concentration of 5 × 10⁸ cfu ml⁻¹, 1 mm acetosyringone, 25-d-old mycelia at 0.2 g ml⁻¹, and co-culture period of 6 d were found to be the most optimal conditions for enhancing the transformation efficiency. The mitotic stability of transferred DNA (T-DNA) was demonstrated by growing eight randomly selected putative transformants in malt extract agar medium for five subcultures. Insertion of T-DNA into the genome of transformants was confirmed by PCR and Southern hybridization. Results showed that 88 % of the mutants contained a single T-DNA insertion. Two of the mutants were observed with different triterpenoid profiles compared with the untransformed cultures. Our results suggest a new functional genomics approach to tag the triterpenoid biosynthesis genes in A. cinnamomea.