In situ transfer of antibiotic resistance genes from transgenic (transplastomic) tobacco plants to bacteria

Interkingdom gene transfer is limited by a combination of physical, biological, and genetic barriers. The results of greenhouse experiments involving transplastomic plants (genetically engineered chloroplast genomes) cocolonized by pathogenic and opportunistic soil bacteria demonstrated that these barriers could be eliminated. The Acinetobacter sp. strain BD413, which is outfitted with homologous sequences to chloroplastic genes, coinfected a transplastomic tobacco plant with Ralstonia solanacearum and was transformed by the plant's transgene (aadA) containing resistance to spectinomycin and streptomycin. However, no transformants were observed when the homologous sequences were omitted from the Acinetobacter sp. strain. Detectable gene transfer from these transgenic plants to bacteria were dependent on gene copy number, bacterial competence, and the presence of homologous sequences. Our data suggest that by selecting plant transgene sequences that are nonhomologous to bacterial sequences, plant biotechnologists could restore the genetic barrier to transgene transfer to bacteria.     

转基因植物中抗生素抗性基因的安全性评价

20世纪80年代以来,植物转基因技术取得突飞猛进的进展。转基因植物大量出现,带来了巨大的经济效益和社会效益。与此同时,转基因植物的释放可能带来的风险也越来越受到人们的重视,抗生素抗性基因是筛选转基因植物的常用基因,其安全性引起了人们的普遍关注,作者就转基因植物中抗生素抗性基因的安全性作一综述。     

Reservoirs of antibiotic resistance genes

A potential concern about the use of antibiotics in animal husbundary is that, as antibiotic resistant bacteria move from the farm into the human diet, they may pass antibiotic resistance genes to bacteria that normally reside in a the human intestinal tract and from there to bacteria that cause human disease (reservoir hypothesis). In this article various approaches to evaluating the risk of agricultural use of antibiotics are assessed critically. In addition, the potential benefits of applying new technology and using new insights from the field of microbial ecology are explained.     

Heat-inducible hygromycin resistance in transgenic tobacco

We have constructed a chimaeric gene consisting of the promoter of the soybean heat shock (hs) gene Gmhsp17,6-L, the coding region of a hygromycin phosphotransferase (hpt) gene, and the termination sequence of the nopaline synthase (nos) gene. This gene fusion was introduced into tobacco by Agrobacterium-mediated gene transfer. Heat-inducible synthesis of mRNA was shown by northern hybridization, and translation of this RNA into a functional protein was indicated by plant growth on hygromycin-containing media in a temperature-dependent fashion. One hour incubation at 40 °C per day, applied for several weeks, was sufficient to express the resistant phenotype in transgenic plants containing the chimaeric hs-hpt gene. These data suggest that the hygromycin resistance gene is functional and faithfully controlled by the soybean hs promoter. The suitability of these transgenic plants for selection of mutations that alter the hs response is discussed.     

耐药基因数据库概述

抗生素是人类历史上的革命性发现,其临床应用挽救了无数患者的生命。但是随着抗生素的广泛使用和滥用,越来越多的病原菌产生了耐药性,甚至出现了具有多重耐药性的"超级细菌"。在人类与病原菌斗争的军备竞赛中,人类即将面临无药可用的境地。针对微生物的耐药性、耐药机制及耐药性传播的研究吸引了众多科研工作者的目光,各种耐药基因数据库以及耐药基因分析工具应运而生。文中对目前耐药领域的基因数据库进行收集整理,从数据库类型、数据特征、耐药基因预测模型以及可分析序列的类型等方面对这些数据库进行论述和介绍。此外,文中对抗金属离子和抗杀菌剂的基因数据库也有所涉及,将为如何选择及使用耐药基因数据库提供参考和帮助。     

Selection of transgenic Xenopus laevis using antibiotic resistance

We previously established lines of transgenic Xenopus laevis expressing green fluorescent protein (GFP) or GFP fusion proteins in the rod photoreceptors of their retinas under control of the X. laevis opsin promoter, which permits easy identification of transgenic animals by fluorescence microscopy. However, GFP tags can alter the properties of fusion partners, and in many circumstances a second selectable marker would be useful. The transgene constructs we used also encode a gene that confers resistance to the antibiotic G418 in cultured mammalian cells. In this study, we show that F2 transgenic offspring of these animals are more resistant to G418 toxicity than their non-transgenic siblings, as are primary transgenic X. laevis. G418 resistance can be used as a selectable marker in transgenic X. laevis, and possibly other aquatic transgenic animals.     

Pathogen resistance of transgenic tobacco plants producing caffeine

Caffeine (1,3,7-trimethylxanthine) is a typical purine alkaloid, and produced by a variety of plants such as coffee and tea. Its physiological function, however, is not completely understood, but chemical defense against pathogens and herbivores, and allelopathic effects against competing plant species have been proposed. Previously, we constructed transgenic tobacco plants, which produced caffeine up to 5 microg per gram fresh weight of leaves, and showed them to repel caterpillars of tobacco cutworms (Spodoptera litura). In the present study, we found that these transgenic plants constitutively expressed defense-related genes encoding pathogenesis-related (PR)-1a and proteinase inhibitor II under non-stressed conditions. We also found that they were highly resistant against pathogens, tobacco mosaic virus and Pseudomonas syringae. Expression of PR-1a and PR-2 was higher in transgenic plants than in wild-type plants during infection. Exogenously applied caffeine to wild-type tobacco leaves exhibited the similar resistant activity. These results suggested that caffeine stimulated endogenous defense system of host plants through directly or indirectly activating gene expression. This assumption is essentially consistent with the idea of chemical defense, in which caffeine may act as one of signaling molecules to activate defense response. It is thus conceivable that the effect of caffeine is bifunctional; direct interference with pest metabolic pathways, and activation of host defense systems.     

抗生素抗性基因在环境中的传播扩散及抗性研究方法

抗生素在医药、畜牧和水产养殖业的大量使用造成了环境中抗性耐药菌和抗性基因日益增加,抗生素抗性基因作为一种新型环境污染物引起人们的广泛关注.本文综述了近年来国内外有关抗生素抗性基因的研究进展,其在水、土壤、空气等环境介质中和动,植物体内的传播扩散,以及开展环境中抗生素抗性基因研究的必要性,重点介绍了有关抗生素抗性（包括抗性细菌和抗性基因）的研究方法,指出抗性基因研究中存在的问题,并对未来的相关研究进行了展望.     

Genetically pyramiding protease-inhibitor genes for dual broad-spectrum resistance against insect and phytopathogens in transgenic tobacco

Protease inhibitors provide a promising means of engineering plant resistance against attack by insects and pathogens. Sporamin (trypsin inhibitor) from sweet potato and CeCPI (phytocystatin) from taro were stacked in a binary vector, using pMSPOA (a modified sporamin promoter) to drive both genes. Transgenic tobacco lines of T0 and T1 generation with varied inhibitory activity against trypsin and papain showed resistance to both insects and phytopathogens. Larvae of Helicoverpa armigera that ingested tobacco leaves either died or showed delayed growth and development relative to control larvae. Transgenic tobacco-overexpressing the stacked genes also exhibited strong resistance against bacterial soft rot disease caused by Erwinia carotovora and damping-off disease caused by Pythium aphanidermatum. Thus, stacking protease-inhibitor genes, driven by the wound and pathogen responsive pMSPOA promoter, is an effective strategy for engineering crops to resistance against insects and phytopathogens.     

Characterisation and transfer of antibiotic resistance genes from enterococci isolated from food

The genetic determinants responsible for the resistances against the antibiotics tetracycline [tet(M), tet(O), tet(S), tet(K) and tet(L)], erythromycin (ermA,B,C; mefA,E; msrA/B; and ereA,B) and chloramphenicol (cat) of 38 antibiotic-resistant Enterococcus faecium and Enterococcus faecalis strains from food were characterised. In addition, the transferability of resistance genes was also assessed using filter mating assays. The tet(L) determinant was the most commonly detected among tetracycline-resistant enterococci (94% of the strains), followed by the tet(M) gene, which occurred in 63.0% of the strains. Tet(K) occurred in 56.0% of the resistant strains, while genes for tet(O) and tet(S) could not be detected. The integrase gene of the Tn916-1545 family of transposons was present in 81.3% of the tetracycline resistant strains, indicating that resistance genes might be transferable by transposons. All chloramphenicol-resistant strains carried a cat gene. 81.8% of the erythromycin-resistant strains carried the ermB gene. Two (9.5%) of the 21 erythromycin-resistant strains, which did not contain ermA,B,C, ereA,B and mphA genes harboured the msrC gene encoding an erythromycin efflux pump, which was confirmed by sequencing the PCR amplicon. In addition, all E. faecium strains contained the msrC gene, but none of the E. faecalis strains. Transfer of the genetic determinants for antibiotic resistance could only be demonstrated in one filter mating experiment, where both the tet(M) and tet(L) genes were transferred from E. faecalis FAIR-E 315 to the E. faecalis OG1X recipient strain. Our results show the presence of various types of resistance genes as well as transposon integrase genes associated with transferable resistances in enterococci, indicating a potential for gene transfer in the food environment.     

Tolerance to atmospheric ozone in transgenic tobacco over‐expressing glutathione synthetase in plastids

A cross between transgenic tobacco ( Nicotiana tabacum L.) plants which over‐expressed either γ‐glutamylcysteine synthetase (cpGSHI) or glutathione synthetase (cpGSHII) in their chloroplasts was used to compare the consequences of over‐expression of components of the glutathione synthetic pathway on tolerance to atmospheric O₃ or paraquat. A high proportion (50%) of those progeny which carried the cpGSHII transgene alone showed tolerance to atmospheric O₃ but not to paraquat. Progeny of an additional two, independent, self‐pollinated primary transgenic lines, which segregated in a Mendelian fashion for the presence of the cpGSHII transgene and therefore included both transformed and non‐transformed (recessive, wild‐type) plants, were also challenged by fumigation with O₃. Again, in both cases, about 50% of the plants expressing the epGSHII transgene were found to be O₃‐tolerant on the basis of reduced ethylene emissions and increased or unchanged total pigment concentrations. However, this tolerance was not due to specific changes in stomatal densities.     

Evolution and ecology of antibiotic resistance genes

A new perspective on the topic of antibiotic resistance is beginning to emerge based on a broader evolutionary and ecological understanding rather than from the traditional boundaries of clinical research of antibiotic-resistant bacterial pathogens. Phylogenetic insights into the evolution and diversity of several antibiotic resistance genes suggest that at least some of these genes have a long evolutionary history of diversification that began well before the 'antibiotic era'. Besides, there is no indication that lateral gene transfer from antibiotic-producing bacteria has played any significant role in shaping the pool of antibiotic resistance genes in clinically relevant and commensal bacteria. Most likely, the primary antibiotic resistance gene pool originated and diversified within the environmental bacterial communities, from which the genes were mobilized and penetrated into taxonomically and ecologically distant bacterial populations, including pathogens. Dissemination and penetration of antibiotic resistance genes from antibiotic producers were less significant and essentially limited to other high G+C bacteria. Besides direct selection by antibiotics, there is a number of other factors that may contribute to dissemination and maintenance of antibiotic resistance genes in bacterial populations.     

Indole acetonitrile-sensitivity of transgenic tobacco containing Arabidopsis thaliana nitrilase genes

 The Arabidopsis thaliana genome has four nitrilase (nitrile aminohydrolase, EC 3.5.5.1) genes (NIT1 to NIT4). These nitrilases catalyze hydrolysis of indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA). Growth of A. thaliana is inhibited by IAN probably due to hydrolysis of IAN to IAA, while the tobacco (Nicotiana tabacum) genome has only NIT4 homologs and is resistant to IAN. In this study, we introduced A. thaliana NIT1 to NIT4 into tobacco. Introduction of NIT1, NIT2 or NIT3 into tobacco conferred growth inhibition by IAN. NIT2 transgenic plants were highly sensitive to IAN, and NIT1 and NIT3 transgenic plants were moderately sensitive. On the other hand, NIT4 transgenic plants were less sensitive to IAN, although some morphological changes in the roots were observed as the wild-type tobacco. These findings suggest that the ability of transgenic tobacco to convert IAN to IAA in vivo is markedly different among transgenes of NIT1 to NIT4. Received: 22 November 1999 / Revision received: 28 January 2000 / Accepted: 4 February 2000     

Myb genes from Hordeum vulgare: tissue-specific expression of chimeric Myb promoter/Gus genes in transgenic tobacco

The structures of the three Myb -related genes Hv1 , Hv5 and Hv33 from barley were determined. They contain a single intron located in the second repeat unit of the Myb -related domain. By analogy to the animal MYB oncoproteins this conserved region of the gene product was shown by filter-binding experiments to exhibit nucleic acid-binding activity. Tobacco plants transgenic for chimeric Myb promoter/ Gus genes express the enzyme in a developmentally controlled and tissue-specific manner. During germination and early stages of plant growth, GUS activity is seen in the root cap and adjacent meristematic tissue. At later stages of plant development, GUS activity is predominantly observed in the shoot apical meristem, the roots and the nodal regions of the stem. Within the stem at stages of secondary growth, Myb promoters are active in defined cell types. In the internode low GUS activity is displayed by the innermost cell layer of the cortex, the starch sheath, that surrounds the vascular cylinder of secondary xylem and phloem tissue, as well as in pith rays originating from vascular cambium initials. In the nodal region Myb promoter-controlled Gus expression is mainly confined to the abaxial starch sheath of the leaf trace, to the branch traces and to internal strands of primary phloem. It is suggested that in addition to their activity in meristematically active plant tissues Myb genes are expressed in conductive tissues that are closely associated with vascular bundles.