Chromatin structure of transferred genes in transgenic plants

The chromatin structure of foreign genes in transgenic tobacco plants was investigated by digestion of nuclei with DNase I and micrococcal nuclease, respectively, followed by restriction and Southern analysis of the digestion products. The results were compared to the differential expression of the different transgenes. Two model systems were used: plants harbouring vector DNA derived from the disarmed vector pGV 3850 and plants harbouring the light-regulated and organ-specifically expressed potato ST-LS1 gene and the cotransferred ncpaline synthase (nos) reporter gene. Our results show that transferred genes are located in DNase l-sensitive domains in all transformants. Slight variations of DNase l-sensitivity of the transferred ST-LS1 constructs in different transformants neither reflected the between-transformant variability of expression nor the organ-specific activity of the transgenes. A deletion event was found responsible for silencing the ST-LS1 gene but not the nos gene in one of the transformants. Whereas no DNase l-hypersensitive sites were found within the 3850-T-DNA and the ST-LS1 gene, one prominent site was mapped to the nos promoter within the ST-LS1 construct in all transformants. Digestion of chromatin harbouring 3850-T-DNA with micrococcal nuclease resulted in a blurred nucleosomal pattern as compared to nucleolar and bulk chromatin, the extent of blurring being independent of the expression of transferred genes. The present results favour the “permissive domain” hypothesis which capitalizes on the chromatin surrounding the integration site as the determining factor for the chromatin structure of incoming alien genes. However, between-transformant variability of expression is not reflected by differential sensitivity to DNase I. Hence, other factors than chromatin structure must be involved in creating “position effects”.     

Technical enzymes produced in transgenic plants

Technical enzymes are used in many industrial applications. Nowadays technical enzymes are often produced in transgenic host organisms. The use of transgenic plants with respect to high level of expression at low costs as a prerequisite for successful commercial production of technical enzymes is discussed. This review summarises recently published examples for production of technical enzymes in plants. In addition, plastid transformation and viral vectors are discussed as methods which might be useful for obtaining high expression level of recombinant proteins in plants.     

转基因植物中报告基因gus的表达及其安全性评价

近十年来,植物转基因技术取得显著进展,许多转基因植物不断问世,其中报告基因ｇｕｓ在植物基因工程和遗传转化中发挥着重要的作用。本文就ｇｕｓ基因的来源、在转基因植物中的表达及其生态风险性与食品安全性作了简要综述。     

Synthesis and accumulation of pea plastocyanin in transgenic tobacco plants

The pea plastocyanin gene in a 3.5 kbp Eco RI fragment of pea nuclear DNA was introduced into tobacco by Agrobacterium-mediated transformation. Regenerated plants contained pea plastocyanin located within the chloroplast thylakoid membrane system. Analysis of seedlings from a self-pollinated transgenic plant containing a single copy of the pea plastocyanin gene indicated that seedlings homozygous for the pea gene contained almost twice as much pea plastocyanin as seedlings hemizygous for the pea gene. Homozygous seedlings contained approximately equal amounts of pea and tobacco plastocyanins. The amount of tobacco plastocyanin in leaves of transgenic plants was unaffected by the expression of the pea plastocyanin gene. The mRNA from the pea gene in tobacco was indistinguishable by northern blotting and S1 nuclease protection from the mRNA found in pea. In both pea and transgenic tobacco, expression of the pea plastocyanin gene was induced by light in leaves but was suppressed in roots. Pea plastocyanin free of contaminating tobacco plastocyanin was purified from transgenic tobacco plants and shown to be indistinguishable from natural pea plastocyanin by N-terminal protein sequencing and ¹H NMR spectroscopy.     

Development of the binary vector pTACAtg1 for stable gene expression in plant: Reduction of gene silencing in transgenic plants carrying the target gene with long flanking sequences

Genetic modification in plants helps us to understand molecular mechanisms underlying on plant fitness and to improve profitable crops. However, in transgenic plants, the value of gene expression often varies among plant populations of distinct lines and among generations of identical individuals. This variation is caused by several reasons, such as differences in the chromosome position, repeated sequences, and copy number of the inserted transgene. Developing a state-of-art technology to avoid the variation of gene expression levels including gene silencing has been awaited. Here, we developed a novel binary plasmid (pTACAtg1) that is based on a transformation-competent artificial chromosome (TAC) vector, harboring long genomic DNA fragments on both sides of the cloning sites. As a case study, we cloned the cauliflower mosaic virus 35S promoter:β-glucuronidase (35S:GUS) gene cassettes into the pTACAtg1, and introduced it with long flanking sequences on the pTACAtg1 into the plants. In isolated transgenic plants, the copy number was reduced and the GUS expressions were detected more stably than those in the control plants carrying the insert without flanking regions. In our result, the reduced copy number of a transgene suppressed variation and silencing of its gene expression. The pTACAtg1 vector will be suitable for the production of stable transformants and for expression analyses of a transgene.     

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。     

Insect-resistant transgenic brinjal plants

A synthetic cry1Ab gene coding for an insecticidal crystal protein (ICP) of Bacillus thuringiensis (Bt) was transferred to brinjal (eggplant) by cocultivating cotyledonary explants with Agrobacterium tumefaciens. Transformant plants resistant to kanamycin were regenerated. Hybridization experiments demonstrated gene integration and mRNA expression. Double-antibody sandwich ELISA analysis revealed Bt toxin protein expression in the transgenic plants. The expression resulted in a significant insecticidal activity of transgenic brinjal fruits against the larvae of fruit borer (Leucinodes orbonalis). The results also demonstrated that a synthetic gene based on monocot codon usage can be expressed in dicotyledonous plants for insect control.     

Protein slot blotting: An easy,rapid and reliable technique to identify the expression of a protein in transgenic plants

A technique based on immunological recognition of a foreign protein in transgenic plants has been developed. It allows a quick and reliable screening of many plant samples, improves the accuracy of the results compared to ELISA and is easier to carry out and more sensitive than a western immunoblot. This technique has also been tested to recognize foreign proteins in rice and tobacco leaf extracts.     

利用转基因植物生产口服疫苗的研究现状

本文概述了利用转基因植物生产口服疫苗的研究现状。分别对转基因植物生产口服疫苗的优点、作用原理、研究方法、已研究的口服疫苗及问题和前景进行了介绍。     

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     

Upstream sequences regulating legumin gene expression in heterologous transgenic plants

Summary We have previously isolated a legumin gene LeB4 from Vicia faba and shown that a 4.7 kb DNA fragment containing the gene leads to seed-specific expression in transgenic tobacco plants. Here we report that the 2.4 kb upstream sequence alone, when fused to either the neomycin phosphotransferase II (nptII) gene or the -glucuronidase (uidA) gene, leads to high enzyme levels in transgenic seeds of both tobacco and Arabidopsis. -Glucuronidase (GUS) activity is especially intense in the cotyledons fading out towards the embryonal root tip, a result confirmed by in situ hybridization. Staining of endosperm cells is consistent in both species. Analysis of a series of promoter deletion mutants fused to the nptII gene and introduced into tobacco plants revealed that about 1 kb of 5-flanking sequence is sufficient for high-level expression but indirect evidence suggests the presence of weak positive regulatory elements further upstream. Deletions leaving only 0.2 kb of upstream sequence reduce enzyme levels to less than 10%. A deletion which destroys the legumin box with its seed protein gene-specific CATGCATG motif has no obvious effects on expression levels.     

Cytokinins and cytokinin-binding sites in transgenic potato plants

The introduction of the gene for cytokinin biosynthesis into the potato genome led to a manifold increase in the level of cytokinins (zeatin and zeatin riboside) in transgenic plants grown in vitro. The high amount of endogenous cytokinins in 20-day-old plants of clone 1339-3A correlated with high cytokinin-binding capacity of ribosomes that is presumably attribute to a cytokinin receptor.     

A rapid and efficient DNA minipreparation suitable for screening transgenic plants

We present a modified method for DNA minipreparation suitable for large-scale screening of transgenic plants. The method is rapid and efficient—one person can prepare DNA from approximately 50 samples per day. The average yield was about 40 μg DNA per 100 mg of fresh tissue, and the A₂₆₀/A₂₈₀ was 1.89–2.03. The total DNA extracted by this method could be used for PCR, restriction enzyme digestion, and Southern blotting.     

Screening of transgenic plants by multiplex PCR

A protocol is described, for the rapid screening of a large number of putative transgenic shoots. Genomic DNA is isolated and screened by PCR. To validate the purity of the DNA, PCR amplification is done with primers homologous to an endogenous gene. Multiplex PCR is used to screen for the transgenic shoots with two sets of primers, one set against the endogenous gene (internal control) and the other set against the gene used in transformation. This protocol has been successfully used on maize, melon, oil-seed rape, pepper, petunia, potato, squash, sugar beet and tobacco.     

Assessing environmental risks of transgenic plants

By the end of the 1980s, a broad consensus had developed that there were potential environmental risks of transgenic plants requiring assessment and that this assessment must be done on a case-by-case basis, taking into account the transgene, recipient organism, intended environment of release, and the frequency and scale of the intended introduction. Since 1990, there have been gradual but substantial changes in the environmental risk assessment process. In this review, we focus on changes in the assessment of risks associated with non-target species and biodiversity, gene flow, and the evolution of resistance. Non-target risk assessment now focuses on risks of transgenic plants to the intended local environment of release. Measurements of gene flow indicate that it occurs at higher rates than believed in the early 1990s, mathematical theory is beginning to clarify expectations of risks associated with gene flow, and management methods are being developed to reduce gene flow and possibly mitigate its effects. Insect pest resistance risks are now managed using a high-dose/refuge or a refuge-only strategy, and the present research focuses on monitoring for resistance and encouraging compliance to requirements. We synthesize previous models for tiering risk assessment and propose a general model for tiering. Future transgenic crops are likely to pose greater challenges for risk assessment, and meeting these challenges will be crucial in developing a scientifically coherent risk assessment framework. Scientific understanding of the factors affecting environmental risk is still nascent, and environmental scientists need to help improve environmental risk assessment.