转基因植物的PCR检测

１９９６—２０００年全球转基因作物的种植面积，由原来的１７０万公顷增到４４２０万公顷，５年间猛增了２５倍。近年来，随着转基因作物的种类和产量的不断增加，其产品的安全性问题，即它对人类健康、环境保护和生态平衡等存在的潜在危害，也越来越多的受到人们的广泛关注。至今，已有２０多个国家已开展了基因工程技术的安全性研究，同时还陆续制定了相关的实验研究、工业化生产和向环境释放等一系列安全准则、条例、法规或法律。１９９８年以来，欧盟规定对含有转基因成分的食品及其它商品必须加注标签。最近，日本、韩国、澳大利亚和…     

转基因植物食品检测技术研究进展

随着转基因植物的迅速发展,转基因植物（GMO）含量大量涌向市场。出于对人类健康的考虑,GMO食品标识制度在世界范围内受到人们普遍关注。不论是对GMO食品贴标签,或是对GMO与非GMO原料的分别输送,GMO原料和食品的检测技术是必不可少的。GMO食品的检测主要有两种方法：一种是以DNA为基础的PCR法,另一种是从蛋白质水平出的ELISA法。比较全面地阐述了这两种检测方法的应用状况,重点介绍了PCR检测法及影响PCR检测方法的因素,作者预测DNA芯片检测法将是未来的发展方向。     

出入境转基因产品及其分子检测现状与展望

随着转基因产品在全球的迅速推广,包括我国在内的很多国家都建立了转基因标识制度。各检验检疫口岸应转基因产品生产企业、食品制造商、消费者等多方面需要,相继开展了转基因产品的检测工作。准确可靠的转基因产品检测技术是各国检疫检疫单位的共同需求。转基因产品的检测主要有两大类方法,一类是DNA水平上的检测,另一类是蛋白质水平上的检测。多个发达国家也相继成立专门机构或部门,负责转基因产品生物检测技术标准化工作。国际上对转基因产品的检测工作有向委托鉴定方向发展的趋势。我们简要综述了出入境转基因产品及其分子检测现状。     

植物体细胞无性系变异的分子基础

TheMoleculBasicofSomaclonalVariationinPlantsZhangChunyiYangHanmin(BiologyDepartmentofLanzhouUniversityLanzhou730000)近年来,随着植物细胞和组织培养技术的迅速发展与广泛应用,不断发现在培养细胞和再生植株中存在着各种不同的变异,其中有些是可以遗传的,这种可遗传的变异被称为体细胞无性系变异[39]．变异的发生有其遗传学基础,具体表现在显微水平上的染色体数目和结构变异与分子水平上的基因突变、碱基修饰、基因扩增或丢失、基因重排以及转座因子的激活而影响核及细胞质基因的表达等等．目前,人们在已经积累的大量有…     

转基因植物检测技术的研究进展

现代植物基因工程使转基因植物及其产品越来越多地进入人们的生活,转基因植物安全性在世界范围内引起了广泛关注,对转基因植物检测技术的需求也越来越紧迫。就转基因植物检测技术的研究进展进行综述,重点介绍以基因和蛋白为目标的检测技术,包括PCR、ELISA和基因芯片技术的最新进展,并对不同方法的优缺点进行对比。此外,提出对特定代谢产物的检测是转基因植物检测的重要组成部分,是以后检测技术的发展趋势之一。最后,以差异蛋白为检测目标,结合研究工作提出基于双向电泳技术的转基因植物检测方法及其产品溯源方案。     

转基因植物中报道基因GUS的活性检测及其应用

GUS基因是目前转基因植物中应用最为广泛的报道基因。GUS基因在转基因植物中的活性检测方法有组织化学定位法、分光光度法、荧光分析法、聚丙烯酰胺凝胶原位分析法等。     

植物转基因成分PCR检测内对照系统的建立

为了建立适用于多种植物的转基因成分PCR检测的内对照系统,本文针对植物叶绿体DNArbcL基因的保守区域,设计了一对扩增片段为433bp的PCR引物。通过对23种植物的PCR扩增表明,该引物不但在4种单子叶植物（大米,玉米,小麦,洋葱）,10种原始花被亚纲的双子叶植物（甘蓝,白菜,大豆,豇豆,花生,胡萝卜,芹菜,菠菜,大麻,棉花）及7种合瓣花亚纲的双子叶植物（圣女果,番茄,辣椒,马铃薯,南瓜,黄瓜,菊苣）中得到了稳定一致的扩增结果,而且在低等的藻类植物（海,经须菜）中也得到了特异性的扩增结果。进一步对扩增片段进行的DNA序列测定与分析表明,扩增片段的变异水平较低,具有较高的保守性。本系统的建立有助于排除PCR检测时的假阴性结果,从而提高检测的准确性,而且能克服现行的“一种植物一种检测内对照”的弊端,有利于提高检测效率,缩短检测周期。     

转基因植物快速检测方法的研究

本试验对转基因植物检测中的DNA提取和PCR扩增程序作了改进。经试验,本研究建立的DNA快速提取法与目前广泛使用的CTAB法相比更为简便,快速和经济,提取的DNA质量主扩增效果无明显差异,可用于多种转基因植物,多种植物组织的DNA提取,利用复合PCR法可在同一反应管中同步检测35N,NOS及CP4－EPSPS基因,明显提高了检测效率。应用本试验建立的DNA快速提取－复合PCR扩增－银染检测技术可在6小时内得出结果,达到了快速,简便,灵敏,可靠的检测目的。     

植物转基因的进展与禾谷类作物转基因的评估

植物转基因技术主要包括病原生物感染系统转基因和理化法直接转基因,这是近五年多来发展极为迅速的领域,我国已有许多研究报道,并有不少综述.本文即在此基础上深入介绍植物转基因的研究进展,及其在禾谷类作物上应用所面临的问题。     

植物转基因的失活与沉默

植物外源基因转化研究揭示有相当比例的植物转基因表现失活与沉默。这一现象涉及转基因重复拷贝之间的异源配对,基因顺序的甲基化,转录后的衰退调控以及插入位点染色质高级结构的改变,并与外源基因的随机整合有关。克服转基因失活的方法包括单拷贝转基因子代筛选,构建细胞核基质支架附着区载体以及采用具有特殊功能的启动子与增强子。转基因失活问题的发生提示人们,外源基因的转化与整合存在一些特有的遗传机制。     

Gene interactions and epigenetic variation in transgenic plants

Unusual gene interactions were observed in several doubly transformed tobacco plants which were obtained following sequential transformation steps using two T-DNAs encoding different selection and screening markers. The expression of T-DNA-I, which encoded kanamycin resistance (Kan^r) and nopaline synthase (NOS), was suppressed in some, but not all, of the double transformants after the introduction of T-DNA-II, which encoded hygromycin resistance (Hyg^r) and octopine synthase (OCS). Double transformants in which T-DNA-I had been inactivated could produce Kan^rNOS⁺ progeny, but these were shown to lack T-DNA-II, thus establishing the role of this T-DNA in the suppression of T-DNA-I. Reversible cytosine methylation of the promoters of T-DNA-I genes was shown to correlate with their activation/inactivation cycle. In this paper we pursue further the questions of the mechanism of suppression of T-DNA-I genes by T-DNA-II, and also the timing and extent of demethylation of T-DNA-I promoters in Kan^r progeny following the loss of T-DNA-II. We propose that the suppression is due to the competition between homologous regions on each T-DNA for binding to nuclear sites with fixed locations. We further suggest that incomplete demethylation patterns of T-DNA-I promoters in Kan^r progeny reflect the existence in the shoot apex meristem of two cell populations, which have either methylated or unmethylated T-DNA-I promoters, respectively. Thus, Kan^r progeny are epigenetic chimeras with respect to the expression of T-DNA-I genes.     

转大麻哈鱼生长激素基因鲤表型性状与体质量的相关性及通径分析

随机选取二龄转大麻哈鱼生长激素基因鲤和对照鲤各30尾,运用相关分析和通径分析的方法研究全长、体长、体高、尾柄高、尾柄长、头长、吻长、眼径、眼间距、体厚10个表型性状对体质量的影响程度,以此确定影响二龄转基因鲤和对照鲤体质量的主要表型参数.相关分析结果表明:转基因鲤和对照鲤的大部分表型性状与体质量间的相关系数均达到极显著水平(P<0.01).通径分析结果显示:体长和体高可以作为预测转基因鲤体质量的主要表型参数,转基因鲤体长对体质量的通径系数为0.572,体高对体质量的通径系数为0.415,体长和体高直接决定体质量;而体厚和头长可以作为预测对照鲤体质量的主要表型参数,对照鲤体厚对体质量的通径系数为0.610,头长对体质量的通径系数为0.377,体厚和头长对体质量具有决定作用.     

Development and deployment of transgenic plants: Biosafety considerations

Summary Recombinant DNA technology has great potential to enhance and extend the advantages of conventional plant breeding, and increase the production and productivity of crops to meet the increasing demand for food and food products in the future. Judicious application of this technology provides opportunities for alleviating some of the major constraints to crop productivity under subsistence farming conditions in the developing countries. Considerable progress has been made in developing strategies for the production and deployment of transgenic crops. However, biosafety concerns have been raised regarding the deployment and release of genetically engineered plants. This debate has divided the farming and consumer communities over acceptability of genetically modified foods. There is a need for a thorough investigation regarding the fate of transgenic plants in the environment, and their interaction with wild relatives and non-target organisms. The production and release of transgenic plants should be based on experience and sound scientific reasoning. The regulatory requirements for deployment of transgenic crops should be streamlined and harmonized, in order to achieve sustainable food production, poverty reduction, and environmental protection in resource-poor countries in the semi-arid tropics.     

First-generation transgenic plants and statistics

The statistical analyses of populations of first-generation transgenic plants are commonly based on mean and variance and generally require a test of normality. Since in many cases the assumptions of normality are not met, analyses can result in erroneous conclusions. Transformation of data to normality, the use of other distributions, or distribution-free statistical tests should then be used to obtain valid conclusions from these populations.     

Development of selection marker-free transgenic potato plants with enhanced tolerance to oxidative stress

A binary vector devoid of a plant selection-marker gene (designated as pSSA-F) was constructed to overcome bio-safety concerns about genetically modified plants. This vector carried chloroplast-targeted superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes under the control of an oxidative stress-inducible(SWPA2) promoter, and was utilized to transform potato (Solanum tuberosum L.). Integration of these foreign genes into transgenic plants was primarily performed via PCR with genomic DNA. Twelve marker-free transgenic lines were obtained by inoculating stem explants. The maximum transformation efficiency was 6.25% and averaged 2.2%. Successful integration of the SOD and APX genes rendered transgenic plants tolerant to methyl viologen-mediated oxidative stress at the leaf-disc and whole-plant levels. Our findings suggest that this technique for developing selection marker-free transgenic plants is feasible and can be employed with other crop species.     

Trans-activation of an artificial dTam3 transposable element in transgenic tobacco plants

In Antirrhinum majus only autonomous Tam3 transposons have been characterized. We investigated whether an artificial dTam3 element, with a deletion in the presumptive transposase coding region, can be trans-activated in tobacco by an activator Tam3 element, which was immobilized by the deletion of one inverted repeat. A phenotypic assay based on restored hygromycin resistance demonstrates that a dTam3 element harbouring a bacterial plasmid can be trans-activated with a low frequency. Molecular analysis confirms that the dTam3 element has been excised from the HPTII marker gene. Reintegration of the dTam3 element into the tobacco genome is detected only in one out of six hygromycin-resistant plants analysed. PCR analysis of empty donor sites shows that excision of the dTam3 element in tobacco results in rearrangements (deletions and additions), that have been shown to be characteristic of Tam3 excision in the original host Antirrhinum majus. This trans-activation assay allowed us to establish that, in contrast to what has been detected in Antirrhinum majus, a periodical temperature shift down to 15°C does not enhance dTam3 transposition in regenerating tobacco calli.     

Selection in a fluctuating environment leads to decreased genetic variation and facilitates the evolution of phenotypic plasticity

Changes in the environment are expected to induce changes in the quantitative genetic variation, which influences the ability of a population to adapt to environmental change. Furthermore, environmental changes are not constant in time, but fluctuate. Here, we investigate the effect of rapid, continuous and/or fluctuating temperature changes in the seed beetle Callosobruchus maculatus, using an evolution experiment followed by a split-brood experiment. In line with expectations, individuals responded in a plastic way and had an overall higher potential to respond to selection after a rapid change in the environment. After selection in an environment with increasing temperature, plasticity remained unchanged (or decreased) and environmental variation decreased, especially when fluctuations were added; these results were unexpected. As expected, the genetic variation decreased after fluctuating selection. Our results suggest that fluctuations in the environment have major impact on the response of a population to environmental change; in a highly variable environment with low predictability, a plastic response might not be beneficial and the response is genetically and environmentally canalized resulting in a low potential to respond to selection and low environmental sensitivity. Interestingly, we found greater variation for phenotypic plasticity after selection, suggesting that the potential for plasticity to evolve is facilitated after exposure to environmental fluctuations. Our study highlights that environmental fluctuations should be considered when investigating the response of a population to environmental change.     

生物工程实验室与转基因植物的安全性评价

本阐述了生物技术和生物安全的科学涵义,对生物工程实验室以及转基因植物潜在的生物安全问题做了详尽的说明,并提出了相应的防范措施和建议,对生物工程特别是转基因植物的前景进行了分析展望。     

转抗虫基因植物生态安全性研究进展

转抗虫基因植物如Bt棉花等已在美国、中国和澳大利亚等国家大规模商业化种植 ,有关转抗虫基因植物潜在的生态风险已引起广泛的关注。该文综述了转抗虫基因植物研究应用现状与安全性研究进展。主要内容包括 :转抗虫基因植物的种类及其对靶标害虫的抗性 ,对非靶标害虫和天敌发生的影响 ,对农田生态系统生物多样性的影响 ,靶标昆虫的抗性治理及转抗虫基因植物的基因漂移等