Expression of correctly processed human growth hormone in seeds of transgenic tobacco plants

Human growth hormone was expressed in transgenic tobacco seeds using the monocot tissue-specific promoter from sorghum -kafirin seed storage protein gene. During tobacco seed ripening, the expressed hormone was directed to the endoplasmic reticulum by a signal peptide from a Coix prolamin and was secreted into the apoplastic space, where it accounted for 0.16% of total soluble seed protein. The expressed hormone has the same amino acid sequence and receptor-binding properties as the native mature hormone.     

利用转基因植物生产药用蛋白研究进展

简要评述了国内外利用转基因植物生产药用蛋白的研究现状、发展趋势,以及转基因植物生产药用蛋白的基本方法、应用研究等。尽管目前植物作为药用蛋白的生物反应器受到诸多因素限制,优点与问题并存,但利用转基因植物生产药用蛋白是植物基因工程研究领域的一个新的发展趋势。     

Normal and lysine-containing zeins are unstable in transgenic tobacco seeds

Chimeric genes composed of the -phaseolin promoter, an -zein coding sequence and its modified versions containing lysine codons, and a -zein polyadenylation signal were inserted into the genome of tobacco by Agrobacterium-mediated transformation. -Zein mRNA levels in the transgenic tobacco seeds 20 days after self-pollination varied between 1.0% and 2.5% of the total mRNA population. At 25 days after pollination the 19 kDa -zein was immunologically detected with a polyclonal antiserum in protein extracts from the seeds of transgenic plants. The transgenic plant with the highest level of zein gene expression had an -zein content that was approximately 0.003% of the total seed protein. The amount of -zein in other transgenic plants varied between 1 × 10^–4% and 1 × 10^–5% of the total seed protein. The differences in the amounts of mRNA and protein did not correlate with the lysine substitutions introduced into the -zein protein. Polysomes translating -zein mRNA isolated from tobacco seeds contained fewer ribosomes than those from maize endosperm, but this did not appear to be the cause of the inefficient protein synthesis. In vivo labelling and immunoprecipitation indicated that newly synthesized -zein was degraded in tobacco seeds with a half-life of less than 1 hour.     

A plant signal sequence enhances the secretion of bacterial ChiA in transgenic tobacco

When the secreted bacterial protein ChiA is expressed in transgenic tobacco, a fraction of the protein is glycosylated and secreted from the plant cells; however most of the protein remains inside the cells. We tested whether the efficiency of secretion could be improved by replacing the bacterial signal sequence with a plant signal sequence. We found the signal sequence and the first two amino acids of the PR1b protein attached to the ChiA mature protein directs complete glycosylation and secretion of the ChiA from plant cells. Glycosylation of this protein is not required for its efficient secretion from plant cells.     

An improved method for the detection and quantification of recombinant protein in transgenic plants

A sensitive method has been developed for the detection of recombinant protein produced as a result of gene transfer into plants. This method is based upon antibody binding, which is then visualized using enhanced chemiluminescence and recorded on x-ray film for long-term storage. The technique is simple, rapid and reliable and can be used to screen large numbers of transgenic plants. Several plant species have been successfully tested in this way for a range of recombinant proteins.     

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.     

转基因植物表达药用蛋白的研究进展

基因工程技术的进步使得转基因植物广泛应用于工业、农业各个领域,尤其在医药制造领域。研究成果表明,转基因植物作为生物反应器在制备药用蛋白,如重组疫苗、重组动物抗体、细胞因子等方面较其他表达系统,如微生物及动物表达系统具有成本低、应用安全等优势,但在工业化技术方面仍存在障碍。     

A trout growth hormone is expressed,correctly folded and partially glycosylated in the leaves but not the seeds of transgenic plants

The growth hormone gene of the rainbow trout (tGH-II) was expressed in leaves of transgenic tobacco plants and seeds of transgenicArabidopsis plants using tissue-specific promoters. Although in the leaves and the seeds comparble amounts of tGH-II mRNA could be detected, the protein could only be identified in the tobacco leaves. Passage of the hormone into the secretory pathway, mediated by the signal sequence of the extracellular tobacco PRI-b (pathogenesis-related) protein, resulted in correct disulphide bridge formation and (partial) glycosylation of the hormone. In contrast, cytoplasmic expression resulted in misfolding and partial breakdown of the protein. The data demonstrate that synthesis, folding and glycosylation of heterologous proteins in plants is dependent both on subcellular location as well as on the tissue or cell type in which the protein is expressed.     

Manipulation of the napin primary structure alters its packaging and deposition in transgenic tobacco (Nicotiana tabacum L.) seeds

Napin is a 2S storage protein found in the seeds of oilseed rape (Brassica napus L.) and related species. Using protein structural prediction programs we have identified a region in the napin protein sequence which forms a `hydrophilic loop' composed of amino acid residues located at the protein surface. Targeting this region, we have constructed two napin chimeric genes containing the coding sequence for the peptide hormone leucine-enkephalin as a topological marker. One version has a single enkephalin sequence of 11 amino acids including linkers and the second contains a tandem repeat of this peptide comprising 22 amino acids, inserted into the napin large subunit. The inserted peptide sequences alter the balance of hydrophilic to hydrophobic amino acids and introduce flexibility into this region of the polypeptide chain. The chimeric genes have been expressed in tobacco plants under the control of the seed-specific napA gene promoter. Analyses indicate that the engineered napin proteins are expressed, transported, post-translationally modified and deposited inside the protein bodies of the transgenic seeds demonstrating that the altered napin proteins behave in a similar fashion to the authentic napin protein. Detailed immunolocalisation studies indicate that the insertion of the peptide sequences has a significant effect on the distribution of the napin proteins within the tobacco seed protein bodies.     

Secretion of a functional single-chain Fv protein in transgenic tobacco plants and cell suspension cultures

A synthetic gene encoding an anti-phytochrome single-chain Fv (scFv) antibody bearing an N-terminal signal peptide has been used to transform tobacco plants. Immunoblot analysis showed that transformed plants accumulate high levels of scFv protein, accounting for up to 0.5% of the total soluble protein fraction, which could be extracted by simple infiltration and centrifugation of leaf tissue. A substantial proportion of the scFv protein extracted in this way was found to possess antigen-binding activity. Callus cell suspension cultures derived from transformed plants secrete functional scFv protein into the surrounding medium. Compared with the levels of scFv protein observed in plants expressing the native scFv gene, the incorporation of an N-terminal signal peptide, to target the scFv to the apoplast, results in elevated accumulation of the protein.     

Pea lectin is correctly processed,stable and active in leaves of transgenic potato plants

A gene encoding the preproprotein of the pea (Pisum sativum) lectin was expressed in transgenic potato plants using a cauliflower mosaic virus (CaMV) 35S promoter or a tobacco ribulose bisphosphate carboxylase small subunit (ssRubisco) promoter. Presence of the pea lectin to levels greater than 1% of total soluble leaf protein was detected by radioimmunoassay (RIA). The pattern of expression derived from the two promoters was established using both RIA and a squash-blot immunolocalisation technique. Western blotting demonstrated that the preproprotein was correctly processed, generating and subunits that assembled to give an isolectin form observed in pea seeds and roots. It was also found that the haemagglutination activity and specificity of pea lectin synthesised in transgenic potato leaves was comparable to purified lectin from pea cotyledons.     

Overexpression of thaumatin-like protein in transgenic tomato plants confers enhanced resistance to Alternaria solani

Abstract

A cDNA encoding thaumatin-like protein (TLP) from rice was cloned into the binary vector pMON410 under the control of the CaMV 35S promoter for Agrobacterium-mediated transformation of tomato. All putative transformants were tested for the integration and expression of the chimeric gene by polymerase chain reaction (PCR) for hygromycin resistance gene (hph) and enzyme-linked immunosorbent assay (ELISA) for TLP respectively. Constitutive, high-level expression of TLP was observed in transgenic plants. The transgenic lines exhibited increased resistance to Alternaria solani, the early blight pathogen compared to non-transgenic tomato plants.     

Biochemical and functional characterization of anti-HIV antibody-ELP fusion proteins from transgenic plants

The stability and recovery of recombinant proteins expressed in plants are improved by fusion to elastin-like peptides (ELPs). In order to test the suitability of ELP for the production of pharmaceutical proteins, transgenic plants were created that individually expressed the light and heavy chains of the broadly neutralizing anti-human immunodeficiency virus type 1 (anti-HIV-1) monoclonal antibody 2F5, which is being evaluated as a microbicide component. The antibody chains were expressed both with and without a C-terminal ELP fusion. Crossing these plants in all combinations resulted in transgenic lines producing the full antibody in four formats, with ELP on either the light or heavy chains, on both or on neither. Characterization of the affinity-purified antibodies by surface plasmon resonance spectroscopy showed that the kinetic binding parameters were identical to those of a Chinese hamster ovary (CHO) cell counterpart lacking ELP. N -Glycan analysis showed that all four derivatives contained predominantly oligo-mannose-type N -glycans and that the ELP fusions had no significant effect on N -glycan structure. It was concluded that ELP fusion to the light chain, heavy chain or both chains of a plant-derived antibody had no adverse affects on protein quality, but had a positive impact on the yield. ELP fusions do not interfere with folding, assembly, trafficking in the secretory pathway or post-translational modification, but enhance stability whilst at the same time simplifying recovery.     

Characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants

Cymbidium mosaic virus (CyMV) is the most prevalent virus infecting orchids. Here, we report the isolation of partial cDNA clones encoding the genomic RNA of CyMV. Like most of the polyadenylated monopartite positive-strand RNA viruses, the open reading frame (ORF) coding for the viral coat protein (CP) is located at the 3 end. The ORF predicts a polypeptide chain of 220 amino acids with a molecular weight of 23 600. Sequence comparison of this ORF to the CP sequences of potato virus X(PVX) and white clover mosaic virus (WCIMV) revealed a strong amino acid homology in the mid-portion of the CP, but the overall homology was low. The CyMV CP gene was placed downstream of a cauliflower mosaic virus 35S promoter and the chimaeric gene was transferred into Nicotiana benthamiana. Transgenic plants expressing the CyMV CP were protected against CyMV infection.     

Paraquat resistance of transgenic tobacco plants over-expressing the Ochrobactrum anthropi pqrA gene

Transgenic tobacco plants over-expressing the Ochrobactrum anthropi pqrA gene, which encodes a membrane transporter mediating resistance to paraquat, were generated. Transgenic plants displayed higher resistance against paraquat than wild-type plants, as estimated by plant viability, ion leakage and chlorophyll loss, but no resistance against other active oxygen generators, such as H2O2 and menadione. Moreover, lower levels of paraquat accumulated in transgenic plants, compared to wild-type plants, indicating that the PqrA protein detoxifies paraquat either via increased efflux or decreased uptake of the herbicide, but not by removing active oxygen species. The results collectively demonstrate that the bacterial paraquat resistance gene, pqrA, can be functionally expressed in plant cells, and utilized for the development of paraquat-resistant crop plants.     

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.     

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