拟南芥LEC同源基因的生物信息学分析

用生物信息学方法对拟南芥叶状子叶(LEC)基因的核苷酸序列以及推导氨基酸序列的组成、功能结构域、亲水性等进行了分析。结果表明,拟南芥的LEC蛋白为位于细胞核中的亲水性不稳定蛋白;通过对LEC蛋白的功能结构域的分析发现,LEC1和L1L蛋白中高度保守的区域即为CBF-NFYB-HMF结构域,LEC2和FUSCA3蛋白中高度保守的区域为B3结构域。     

Floral transition mutants in Arabidopsis

An inventory of genetic differences in flowering time in Arabidopsis is presented and discussed. Many genes influence the transition to flowering in a quantitative way. Two groups of mutants and natural variants can be distinguished: those that are responsive to environmental factors and those that are less responsive or unresponsive. It is possible that all late/early-flowering mutants isolated to date carry a mutation with an effect, either promotive or repressive, on a floral repressor. The interaction between light perception and flowering has been studied by analysis of phytochrome- and cryptochrome-deficient mutants, which showed that phyA and probably also cryptochrome have a promotive role in flowering, whereas phyB and other stable phytochromes have an inhibitory role. A circadian rhythm is important in establishing daylength sensitivity, as was shown by the phenotype of the elf 3 mutants.     

Analysis of Tag1-like elements in Arabidopsis thaliana and their distribution in other plants.

Analysis of genomic DNA of Arabidopsis Columbia (Col.) ecotype using a transposon Tag1-specific primer showed the presence of Tag1 homologues which was confirmed by Southern hybridization with a Tag1 probe. Further analysis showed that the homologue, 0.75 kb in length, had inverted repeats at both ends, 8-bp duplicated sequences at the site at which it is located and about 80% homology with Tag1, and was randomly distributed in the Arabidopsis genome. Based on these results, we concluded that these elements are non-autonomous variants of Tag1 and we termed this element sTag1. Using the polymerase chain reaction fragment hybridization technique, we found the distribution of such homologues in other plant species.     

Identification of protein-coding regions in Arabidopsis thaliana genome based on quadratic discriminant analysis

A new method (MZEF) for predicting internal coding exons in genomic DNA sequences has been developed. This method is based on a prediction algorithm that uses the quadratic discriminant function for multivariate statistical pattern recognition. With improved feature measures, an Arabidopsis thaliana-specific implementation of MZEF is completed and made available to the plant genome community.     

拟南芥抗盐突变体的RAPD分析

以筛选得到可以稳定遗传的抗盐单基因突变体2^#和15^#以及野生型拟南芥为材料进行RAPD分析,150条引物中有3条引物在突变体之间扩增出多态性,不仅证明了DNA水平突变的发生,而且表明它们之间遗传背景相似,是一系列抗盐性不同的近似等位基因系。1条引物在突变体的扩增产物比在野生型的扩增产物多出一个大小约为1200bp的片段,初步认为该片段与抗盐的主效基因有关。     

Late embryo-defective mutants of Arabidopsis

The embryo-defective (emb) mutants of Arabidopsis constitute a large and diverse group of mutants disrupted in a broad range of embryonic processes, including morphogonesis, cell differentiation, and maturation programs. This report describes a subset of these mutants, the late embryo defectives, which develop beyond the globular stage of embryogenesis but fail to complete normal morphogenesis. A representative sample of 12 late mutants was chosen for this study, patterns of morphogenesis were characterized, the germination potential of mutant seeds was investigated, and additional mutant alleles within the collection were identified. Morphological defects in mutant embryos became apparent during the heart stage of development, when embryos normally begin the rapid cell division and expansion required for the completion of morphogenesis. Despite their morphological abnormalities, mutant embryos often germinated from dry seed, demonstrating that genetic programs required for the establishment of desiccation tolerance remained intact. Mutant seedlings displayed a wide range of developmental abnormalities, including altered morphology, lack of pigmentation, dwarfism, and disorganized vegetative growth. One late mutant was found to be allelic to an early embryo defective that arrests at the globular stage. These results suggest that a number of late EMB genes encode basic cellular and metabolic functions needed for cell division, enlargement, and embryonic growth. The rapid growth and metabolic changes that occur at the heart stage may present a barrier to normal development in the late mutants, resulting in altered embryo morphology and other developmental defects. It is proposed that many Arabidopsis mutants with abnormal embryo and seedling morphology are not defective in the regulation of pattern formation or morphogenesis, but rather in fundamental physiological and cellular processes required for the completion of normal growth and development. © 1995 Wiley-Liss, Inc.     

In situ RNA hybridization using Technovit resin in Arabidopsis thaliana

A protocol is described for RNA in situ hybridization using thin sections prepared by Technovit resin. Technovit is a widely used resin for histological examinations. Since it does not require time-consuming processes such as removal of the resin and can be performed without high temperature treatment, a high resolution of sections could be possible compared to other resins and paraffin. Thin sections (approximately 4 m) were made from inflorescences of Arabidopsis thaliana embedded in Technovit 8100 resin, and in situ hybridization was performed using the protocol described in this article. Hybridization signals were observed using LEAFY and other genes as probes, showing that this resin can be used for in situ analysis. In our experiments, the most important factor for a successful in situ hybridization pattern was to optimize the RNase A concentration after hybridization. We routinely used RNase A at a concentration of 2–5 ng/ml, a concentration much lower than that used for paraffin embedding method. Thus, the use of the Technovit resin for plant tissue embedding results in a faster protocol and greater quality than allowed by paraffin sections.     

拟南芥活性氧不敏感型突变体的筛选与特性分析

采用 EMS化学诱变方法与 H2 O2 氧化胁迫选择 ,以根在重力作用下的弯曲生长为指标 ,筛选得到拟南芥活性氧不敏感型突变体。对突变体杂交后代遗传分析表明 ,突变株对活性氧不敏感性状为隐性单基因突变所致 ;生理生化分析表明突变体对 H2 O2 有很强的抗性 ,表现为气孔开度对 H2 O2 不敏感和 H2 O2 胁迫时较低的膜脂过氧化水平。运用 L SCM技术并结合 H2 O2 荧光探针 H2 DCFDA检测外源 ABA诱导保卫细胞内产生 H2 O2 的情况 ,结果显示突变体体内荧光强度比对照低 ,暗示了突变体体内消除 H2 O2 的能力可能有所提高 ,增强了植株对氧化胁迫的抗性。拟南芥活性氧不敏感突变体的筛选 ,不仅为人们深入研究活性氧在细胞内的作用提供良好的实验材料 ,而且还将大大加深人们对信号转导途径的再认识     

Analysis of leaf proteins in late flowering mutants of Arabidopsis thaliana

Late flowering monogenic mutants of Arabidopsis thaliana (L.) Heynh. at the loci co, gi, fca, fve, fwa, fha, fpa, fy and their corresponding wild type, Landsberg erecta , were analysed by two-dimensional gel electrophoresis. All plants were grown under continuous light and proteins were extracted from leaves of the same age (20-day-old). The polypeptide patterns of the mutants at the loci co, gi, fca, fve, fwa, fha, fpa , and Landsberg erecta were identical. The mutant at the fy locus showed a qualitative difference with Landsberg erecta . Crosses were made between this line and the wild type Landsberg erecta . F2 plants, resulting from autopollination of the hybrid, were analysed and showed no cosegregation between the observed protein and the flowering phenotype, indicating that these two lines differ by more than a single mutation.     

Variation in dynamics of phytochrome A in Arabidopsis ecotypes and mutants

Phytochromes are photoreceptors in plants which can exist in two different conformations: the red light‐absorbing form (P_r) and the far‐red light‐absorbing form (P_fr), depending on the light quality. The P_fr form is the physiologically active conformation. To attenuate the P_fr signal for phytochrome A (phyA), at least two different mechanisms exist: destruction of the molecule and dark reversion. Destruction is an active process leading to the degradation of P_fr. Dark reversion is the light‐independent conversion of physiologically active P_fr into inactive P_r. Here, we show that dark reversion is not only an intrinsic property of the phytochrome molecule but is modulated by cellular components. Furthermore, we demonstrate that dark reversion of phyA may be observed in Arabidopsis ecotype RLD but not in other Arabidopsis ecotypes. For the first time, we have identified mutants with altered dark reversion and destruction in a set of previously isolated loss of function PHYA alleles (Xu et al. Plant Cell 1995, 7, 1433–1443). Therefore, the dynamics of the phytochrome molecule itself need to be considered during the characterization of signal transduction mutants.     

9个拟南芥凝集素类受体激酶基因功能的初步研究

植物凝集素类受体激酶(LecRLK)在植物防御、应答多种生物与非生物胁迫、参与激素调控和生长发育中具有重要的作用。但是该家族大部分成员的功能尚不明了。本文对LecRLK家族中的9个成员进行了生物信息学和相关分子遗传学的初步研究。启动子元件分析结果表明,9个成员均具有多个光和逆境响应元件,暗示其可能参与光信号以及非生物胁迫信号途径的调控。基因芯片及实时定量PCR结果显示,9个LecRLK基因具有明显的时空表达特异性,说明其可能在植物不同生长发育阶段及组织部位发挥着重要调控作用。利用三引物法对9个基因的突变体进行了筛选,最终得到14个纯合缺失突变体材料。上述结果为后续深入开展这些基因的生理功能研究提供参考数据。     

Classification of Genes Differentially Expressed during Water-deficit Stress in Arabidopsis thaliana: an Analysis using Microarray and Differential Expression Data

Many changes in gene expression occur in response to water-deficitstress. A challenge is to determine which changes support plantadaptation to conditions of reduced soil water content and whichoccur in response to lesions in metabolic and cellular functions.Microarray methods are being employed to catalogue all of thechanges in gene expression that occur in response to specificwater-deficit conditions. Although these methods do not measurethe amount or activities of specific proteins that functionin the water-deficit response, they do target specific biochemicaland cellular events that should be detailed in further work.Potential functions of approx. 130 genes of Arabidopsis thalianathat have been shown to be up-regulated are tabulated here.These point to signalling events, detoxification and other functionsinvolved in the cellular response to water-deficit stress. Asmicroarray techniques are refined, plant stress biologists willbe able to characterize changes in gene expression within thewhole genome in specific organs and tissues subjected to differentlevels of water-deficit stress.     

Islands of co-expressed neighbouring genes in Arabidopsis thaliana suggest higher-order chromosome domains

Biochemical and cytogenetic experiments have led to the hypothesis that eukaryotic chromatin is organized into a series of distinct domains that are functionally independent. Two expectations of this hypothesis are: (i) adjacent genes are more frequently co-expressed than is expected by chance; and (ii) co-expressed neighbouring genes are often functionally related. Here we report that over 10% of Arabidopsis thaliana genes are within large, co-expressed chromosomal regions. Two per cent (497/22,520) of genes are highly co-expressed (r > 0.7), about five times the number expected by chance. These genes fall into 226 groups distributed across the genome, and each group typically contains two to three genes. Among the highly co-expressed groups, 40% (91/226) have genes with high amino acid sequence similarity. Nonetheless, duplicate genes alone do not explain the observed levels of co-expression. Co-expressed, non-homologous genes are transcribed in parallel, share functions, and lie close together more frequently than expected. Our results show that the A. thaliana genome contains domains of gene expression. Small domains have highly co-expressed genes that often share functional and sequence similarity and are probably co-regulated by nearby regulatory sequences. Genes within large, significantly correlated groups are typically co-regulated at a low level, suggesting the presence of large chromosomal domains.     

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.     

拟南芥铵超敏感突变体amosd和vtc1对外源铵的响应

分析了不同外源铵浓度(0、1、5、10、20mmol.L-1)处理下,2个铵超敏感突变体amosd和vtc1对于外源铵处理的响应差异。结果表明,尽管amosd和vtc1都表现为对外源铵超敏感,但二者对外源铵处理浓度的敏感性上存在差异。随着外源铵浓度的增加,vtc1比amosd先表现出铵中毒症状,更高浓度(20mmol·L-1)铵处理时amosd受到的毒害程度表现更加严重,AMOSD遗传位点的缺失容易导致植物出现铵毒害死亡。其次,二者在遭受外源铵胁迫时表现的最敏感部位有所不同,主要的毒害特征上存在差异,amosd在铵胁迫下首先表现在叶片尤其是新叶的发育受阻,而vtc1则主要表现在根部尤其是主根的伸长受阻。通过分区供应实验证明,amosd主要对于地上部供铵处理超敏感,对根部供铵处理不表现超敏感特性;而vtc1则相反,对根部供铵处理超敏感,对地上部供铵不表现超敏感特性。由此可见,amosd和vtc1这2个铵超敏感突变体在拟南芥铵毒害范围和部位上存在较大差异,与vtc1有所不同,amosd是一个叶源铵超敏感型突变体。在农业机械化叶面喷施施肥日益增加和环境铵沉降日益严重的当下,叶源型铵超敏感突变体amosd的获得为揭示植物地上部铵毒害机制提供了一个理想的遗传材料,对系统全面认识植物铵毒害机制,提高作物耐铵性状具有十分重要的意义。     

1,135 ionomes reveal the global pattern of leaf and seed mineral nutrient and trace element diversity in Arabidopsis thaliana

Soil is a heterogeneous reservoir of essential elements needed for plant growth and development. Plants have evolved mechanisms to balance their nutritional needs based on availability of nutrients. This has led to genetically based variation in the elemental composition, the ‘ionome’, of plants, both within and between species. We explore this natural variation using a panel of wild-collected, geographically widespread Arabidopsis thaliana accessions from the 1001 Genomes Project including over 1,135 accessions, and the 19 parental accessions of the Multi-parent Advanced Generation Inter-Cross (MAGIC) panel, all with full-genome sequences available. We present an experimental design pipeline for high-throughput ionomic screenings and analyses with improved normalisation procedures to account for errors and variability in conditions often encountered in large-scale, high-throughput data collection. We report quantification of the complete leaf and seed ionome of the entire collection using this pipeline and a digital tool, Ion Explorer , to interact with the dataset. We describe the pattern of natural ionomic variation across the A. thaliana species and identify several accessions with extreme ionomic profiles. It forms a valuable resource for exploratory genetic mapping studies to identify genes underlying natural variation in leaf and seed ionome and genetic adaptation of plants to soil conditions.     

拟南芥SDIR1基因转录的选择性剪接

采用PCR及RT-PCR法分别克隆了拟南芥SDIR1基因的DNA和cDNA序列。根据序列比对分析结果,发现了3种不同的转录本,提示SDIR1基因的转录中存在选择性剪接。3种转录本的长度分别为822bp、691bp和666bp,依次命名为：SDIR1-822、SDIR1-691、SDIR1-666。与SDIR1基因的DNA序列及已报道的SDIR1cDNA序列比较,除转录本SDIR1-822包含了完整的编码序列外,其余2种转录本的编码序列都存在不同长度的缺失。其中,SDIR1-691缺失了131bp的片段：第2外显子3′端缺失33bp,第3外显子53bp全部缺失,第4外显子5′端缺失45bp;转录本SDIR1-666缺失了156bp的片段：第3外显子3′端缺失18bp,第4外显子5′端缺失138bp。进而随机挑取101个克隆子对三种转录本的表达比例进行初步分析,结果表明3种分子的比值为SDIR1-822：SDIR1-691：SDIR1-666=26.00：1.33：1.00,反映出SDIR1基因不同转录本在拟南芥中的相对表达量。