Expression of the Zinnia TED3 promoter in developing tracheary elements of transgenic Arabidopsis

To determine the regulatory mechanism of gene expression in the early stages of tracheary element (TE) differentiation, we isolated and characterized a genomic fragment of TED3 whose mRNA is expressed preferentially in differentiating TEs 12–24 h before morphological changes in the in vitro Zinnia system. Transgenic Arabidopsis plants with a chimeric gene of the 537 bp TED3 promoter and the -glucuronidase (GUS) reporter gene indicated the strong expression of the GUS gene by the TED3 promoter in TEs, in particular in immature TEs as well as stipules and trichomes. GUS expression driven by the promoter was also induced in callus, in which GUS activity was localized in immature TEs and slender cells around TEs that may be TE precursor cells. The TED3 promoter was not significantly activated by wounding. This pattern of expression differed clearly from that of other vascular tissue-related genes such as PAL, 4CL, and GRP1.8. The nature of TED3 promoter enables us to use it to monitor TE differentiation in tissue and to introduce foreign genes preferentially into immature TE.     

百日草游离叶肉细胞导管分子分化过程中过氧化物酶的催化特性

百日草游离对肉细胞随着导管分子的分化,木质素含量逐渐增加;ＰＯⅠ（可溶性ＰＯ）、ＰＯⅡ（与细胞壁离子型结合的ＰＯ）和ＰＯⅢ（与细胞壁共价结合的ＰＯ）活性增加,并分别对底物愈创木酚、丁子香酚和咖啡酸（含阿魏酸）有较大的亲和力;抑制剂对ＰＯ活性抑制的动力学特性表明．间苯三酚为竞争性抑制剂,硫酸亚铁铵和二硫苏糖醇是非竞争性抑制剂。     

In vivo electroporation for genetic manipulations of whole Hydra polyps

In vivo electroporation is used to study gene regulation and gene function in the freshwater polyp Hydra. Although this approach has been used successfully by several investigators, efficacy and handling continue to present a problem. Here we show technical aspects of in vivo electroporation for introducing fluorescent dyes, plasmid DNA and double stranded RNA into Hydra polyps. We describe the fundamentals of the electroporation delivery system, discuss recent studies where this approach has been used successfully, compare it to alternative transfection methods such as lipofection, and identify future directions.     

RNA干涉技术(RNAi)应用研究进展

RNA干涉是通过双链RNA的介导特异性地降解相应序列的mRNA,从而导致转录后水平的基因沉默,这一过程在拟南芥、线虫和真菌等多种模式植物中发现。RNAi是研究多种生物基因功能的有效手段,本文综述了RNA干涉的分子机理及其应用研究进展。     

L-α-Aminooxy-β-phenylpropionic acid inhibits lignification but not the differentiation to tracheary elements of isolated mesophyll cells of Zinnia elegans

The influence of L-α-aminooxy-β-phenylpropionic acid (AOPP), an inhibitor of L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), and thus of lignin formation, on the differentiation of tracheary elements from isolated mesophyll cells of Zinnia elegans L. cv. Canary Bird was investigated. At low concentrations of AOPP (5–25 μ,M) lignification of differentiating cells was almost completely prevented whereas number of differentiating cells, viability of the cells, fresh and dry weights, and the packed cell volume were higher than corresponding parameters in control cultures. At higher concentrations of AOPP (50–75 μ M ) the formation of tracheary elements was inhibited but division, elongation and expansion of cells were still observed. Cells cultured for 96 h in the presence of 100 μ M AOPP were morphologically similar to cells at 12 h of culture, the time at which AOPP was added. At concentrations of AOPP that did not inhibit differentiation, AOPP caused an increase in the amounts of uronic acid and total carbohydrate (per unit volume of cell suspension) in the extracellular polysaccharide fraction and in the total cell wall fraction, although these parameters were not significantly different from control values when expressed on a dry weight basis. AOPP caused the release of polysaccharides which contained xylose into the medium when added before the onset of visible differentiation and the release of polysaccharides which contained glucose when added at the time when the formation of the secondary cell wall thickenings took place. The results indicate that AOPP at low concentrations specifically inhibits the formation of lignin without adversely affecting the synthesis of cell wall polysaccharides, although the proper integration of these compounds into the wall may be disturbed. O-Benzylhydroxyla-mine, on the other hand, did not prove to be a useful agent to affect lignin synthesis in differentiating Zinnia cells.     

RNA干涉技术

RNA干涉(RNAi)技术是利用一些小的双链RNA来高效、特异地阻断体内特定基因的表达,并促使mRNA降解,从而诱使细胞表现出特定基因缺失的表型。本从RNAi技术的历史、作用机制、研究策略、研究现状及应用前景等几个方面进行了综述,预测RNAi将会给基因治疗的发展带来新的希望。     

Involvement of local intercellular communication in the differentiation of zinnia mesophyll cells into tracheary elements

The transdifferentiation of isolated mesophyll cells of zinnia (Zinnia elegans L.) into tracheary elements (TEs) has been well studied as a model of plant cell differentiation. In order to investigate intercellular communication in this phenomenon, two types of culture method were developed, in which mesophyll cells were embedded in a thin sheet of agarose gel and cultured on solid medium, or embedded in microbeads of agarose gel and cultured in liquid medium. A statistical analysis of the two-dimensional distribution of TEs in the thin-sheet cultures demonstrated their aggregation. In the microbead cultures, the frequency of TE differentiation was shown to depend on the local cell density (the cell density in each microbead): TE differentiation required local cell densities of more than 10⁵ cells ml⁻¹. These results suggest that TE differentiation involves cell-cell communication mediated by a locally acting diffusible factor. This presumptive factor was characterized by applying a modified version of the sheet culture, which used two sheets of different cell densities, a low-density sheet and a high-density sheet. Differentiation of TEs in the former could be induced only by bringing it into contact with the latter. Insertion of a 25-kDa-cutoff membrane between the high-density and low-density sheets severely suppressed such induction of TEs in the low-density sheet while a 300-kDa-cutoff membrane suppressed induction only slightly. Insertion of agarose sheets containing immobilized pronase E or trypsin also interfered with the induction of TEs in the low-density sheets. Thus, a proteinaceous macromolecule of 25–300 kDa in molecular weight was assumed to mediate the local intercellular communication required for TE differentiation. This substance was designated “xylogen” with reference to its xylogenic activity. The time of requirement for xylogen during TE differentiation was assessed by experiments in which cells in the low-density sheet were separated from xylogen produced in the high-density sheet at various times by insertion of a 25-kDa-cutoff membrane between the two sheets, and was estimated to be from the 36th hour to the 60th hour of culture (12–36 h before visible thickening of secondary cell walls of TEs). Received: 13 July 2000 / Accepted: 4 October 2000     

A Transient RNA Interference Assay System Using Arabidopsis Protoplasts

Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana.     

RNA-dependent RNA polymerase 6 is required for efficient hpRNA-induced gene silencing in plants

In plants, transgenes with inverted repeats are used to induce efficient RNA silencing, which is also frequently induced by highly transcribed sense transgenes. RNA silencing induced by sense transgenes is dependent on RNA-dependent RNA polymerase 6 (RDR6), which converts single-stranded (ss) RNA into double-stranded (ds) RNA. By contrast, it has been proposed that RNA silencing induced by self-complementary hairpin RNA (hpRNA) does not require RDR6, because the hpRNA can directly fold back on itself to form dsRNA. However, it is unclear whether RDR6 plays a role in hpRNA-induced RNA silencing by amplifying dsRNA to spread RNA silencing within the plant. To address the efficiency of hpRNA-induced RNA silencing in the presence or absence of RDR6, Wild type (WT, Col-0) and rdr6-11 Arabidopsis thaliana lines expressing green fluorescent protein (GFP) were generated and transformed with a GFP-RNA interference (RNAi) construct. Whereas most GFP-RNAi-transformed WT lines exhibited almost complete silencing of GFP expression in the T1 generation, various levels of GFP expression remained among the GFP-RNAi-transformed rdr6-11 lines. Homozygous expression of GFP-RNAi in the T3 generation was not sufficient to induce complete GFP silencing in several rdr6-11 lines. Our results indicate that RDR6 is required for efficient hpRNA-induced RNA silencing in plants.     

RNAi技术在昆虫功能基因研究中的应用进展

RNA干扰（RNA interference,RNAi）是指外源或内源的双链RNA（dsRNA）特异性地引起基因表达沉默的现象,它作为一种有效的工具用来产生转录后沉默,从而抑制特定基因的表达,成为基因功能研究的一种新方法,除了在模式昆虫如果蝇Drosophila中广泛应用之外,也在非模式昆虫中得到成功应用。近年来,RNAi技术在导入方法和基因功能分析方面都取得了飞速发展,且与转基因技术相结合成功应用于害虫防治领域。本文综述了RNAi技术在导入方法、昆虫功能基因组功能分析及害虫防治等领域新近的研究成果,并展望了该技术的应用前景。