Agrobacterium Ti plasmids as vectors for plant genetic engineering.
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In many plant RNA viruses, Domains 1, 2 and 3 are conserved in replicase proteins. In order to examine the interference of viral replication by the Domain 1 sequence, we generated transgenic plants transformed with DNA corresponding to the Domain 1 sequence of the TMV 126 kDa protein. This DNA sequence includes the TMV RNA from nucleotides 1 to 2,149, which comprises both the 5'-untranslated and methyl transferase region. The transgenic plants obtained showed complete resistance to TMV infection. The presence of the Domain 1 sequence in the plants completely prevented local necrosis in Nicotiana tabacum cv. Xanthi nc, and any systemic development of symptoms in Nicotiana tabacum Xanthi upon TMV inoculation. Most transgenic plants sustained the conferred resistance even under TMV inoculum concentrations up to as high as 1,000 microg/ml. To detect any accumulation of TMV coat protein or viral RNA in infected transgenic plants, immunochemical tests and Northern blot analyses were carried out. Neither viral RNA or coat protein was detectable in the systemic leaves of the completely resistant transgenic plants, whereas they were accumulated in large quantities in all of the control plants. Because of the conservation of Domain 1 in many plant RNA viruses, the acquisition of resistance to virus infection using the Domain 1 sequence appears to be a very effective strategy for breeding of viral resistant plants.
In plant seeds, most of the phosphate is in the form of phytic acid. Phytic acid is largely indigestible by monogastric animals and is the single most important factor hindering the uptake of a range of minerals. Engineering crop plants to produce a heterologous phytase improves phosphate bioavailability and reduces phytic acid excretion. This reduces the phosphate load on agricultural ecosystems and thereby alleviates eutrophication of the aquatic environment. Improved phosphate availability also reduces the need to add inorganic phosphate, a non-renewable resource. Iron and zinc uptake might be improved, which is significant for human nutrition in developing countries.
The mortality of 461 workers who were employed 10 or more years in a Midwest engine and construction equipment plant was examined, using the method of proportional mortality ratios. Both state and national deaths were used as the standard population. Major exposures in this plant included solvents, cutting oils, and metal fumes and dusts. However, precise exposure data were not available. Among white males, no significant deviations from expected deaths were found. Among black males, significant excess deaths were found for all malignant neoplasms combined, for cancer of the pancreas, and for non-Hodgkin's lymphomas. Proportional cancer mortality ratios produced similar results, although the excess of pancreatic cancer in blacks was only significant among those with 20 or more years of service. Although complete occupational histories were not available, these results may provide hypotheses for future studies of workers in heavy machinery production.
Activation tagging is a new method for isolation and functional identification. It can generate dominant gain-of-function mutants by overexpression of a particular endogenous gene. Due to this special characteristics of activation tagging,this method has been a powerful tool for new gene discovery and gene functional analysis. This paper reviewed the principle and study conditions of activation tagging,as well as its use in plant genetic engineering.
Techniques for plant transformation have been developed to such an extent that a number of foreign genes are currently being introduced into transgenic plants. Tobacco plants that produce monoclonal antibodies are of interest, because in addition to synthesis of two gene products (i.e. the heavy and light chains), the two polypeptides need to be assembled correctly, in order to result in a functional antibody. The studies on a catalytic antibody suggest that this is the case, and that the antibody functions identically to the native murine-derived antibody. The only difference observed was in the glycosylation of the heavy chain. Further transgenic plants are being generated to produce monoclonal antibodies that may be used therapeutically (and are therefore required in large quantities), or to provide disease resistance in plants. In addition, the ability of plants to assemble antibody complexes is being investigated further, to study the possibility of generating secretory IgA, which consists of heavy and light chains as well as two additional polypeptide units.
Very long-chain polyunsaturated fatty acids (VLCPUFAs) are essential for human health and well-being. However, the current sources of these valuable compounds are limited and may not be sustainable in the long term. Recently, considerable progress has been made in identifying genes involved in the biosynthesis of VLCPUFAs. The co-expression of these genes in model systems such as plant embryos or yeast provided many valuable insights into the mechanisms of VLCPUFA synthesis. The recent successful reconstitution of pathways leading to the synthesis of arachidonic acid, eicosapentaenoic acid and finally docosahexaenoic acid in oil-seed plants indicates the feasibility of using transgenic crops as alternative sources of VLCPUFAs. The various approaches used to attain these results and the specific constraints associated with each approach are discussed.
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Plant transformation has had a deep impact on several aspects of basic and applied research. Genetic transformation has offered new opportunities compared to traditional breeding practises since it allows the integration into a host genome of specific sequences leading to a strong reduction of the casualness of gene transfer. One of the first target areas was plant protection against pests, pathogens and environmental stresses while the recent plant engineering programs are aimed at increasing food quality, in particular at increasing nutritional characteristics of food crops. Moreover, transgenic plants, tissue or cell cultures represent an attractive biological system for producing heterologous proteins since they offer economic and qualitative benefits. High yield production can be obtained and large-scale commercial production will take advantage of the existing infrastructure for crop cultivation, processing and storage. There are also qualitative benefits since protein synthesis secretion and post-translational modifications are similar in plants and animal cells. There are no human viral pathogens harboured by plants: thus, especially for pharmaceuticals, plants represent the safer production system. Plant transformation has become an essential instrument also for basic research, in particular for the functional characterisation of genes identified by sequencing of whole genomes. Large collections of insertion mutants have been obtained in the model plant Arabidopsis to provide a high level of genome saturation that means 95% chance of inactivating any gene at least once. To instil greater public confidence in modern plant biotechnology recent advances have already been made to overcome the potential risks for human health and environment.
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Over the past few years, there has been a growing realization that metabolic pathways must be studied in the context of the whole cell rather than at the single pathway level, and that even the simplest modifications can send ripples throughout the entire system. Attention has therefore shifted away from reductionist, single-gene engineering strategies and towards more complex approaches involving the simultaneous overexpression and/or suppression of multiple genes. The use of regulatory factors to control the abundance or activity of several enzymes is also becoming more widespread. In combination with emerging methods to model metabolic pathways, this should facilitate the enhanced production of natural products and the synthesis of novel materials in a predictable and useful manner.
The use of transgenic plants for the production of recombinant proteins is not a universal solution for all proteins. The choice of this expression system depends very much on the type of protein and its applications. Many proteins will best be made by conventional microbial fermentation, similarly, we are already identifying proteins where plants represent the only practical option for one reason or another. It will be important to understand better the cellular mechanisms of protein folding, assembly and processing in plants, in order to maximise the potential of transgenic plants as a protein production system. One of the main advantages that plants offer is that they are higher eukaryotic organisms with an endomembrane system. Therefore, they fold and assemble recombinant proteins using protein chaperones that are homologous to those in mammalian cells, and they perform post-translational modifications. This allows, for example, the expression of monoclonal antibodies, first described in 1989, as well as a range of other types of immunoglobulin molecules and multimeric complexes.
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Alkaloids purified from plants provide many pharmacologically active compounds, including leading chemotherapy drugs. As is generally true of secondary metabolites, overall productivity is low, making commercial production expensive. Alternative production methods remain impractical, leaving the plant as the best source for these valuable chemicals. Recently, significant progress in characterizing the biosynthetic pathways leading to various alkaloids has been made, and a number of relevant genes have been cloned. Metabolic engineering employing such genes provides a promising technology for improved productivity in plant cell cultures, plant tissue cultures, or intact plants. In exploring solutions though, metabolic engineers must be careful to recognize the limitations inherent in designing plant systems.
In an important recent paper Kristensen et al. address a question of fundamental importance in plant biotechnology: how are metabolic pathways affected upon introduction of a transgene? Analysis of the transcriptome and metabolome of Arabidopsis thaliana engineered to produce the cyanogenic glucoside dhurrin demonstrated that plants can be tailored in a rational manner with marginal inadvertent effects.
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In many cases, multiple pathway enzymes need to be upregulated to produce a significant yield of a desired product. Technical advances in simultaneously manipulating multiple steps in plant metabolic pathways include the use of transcription factors, such as MYB12. By upregulating the genes of an entire pathway, these factors can greatly simplify multienzyme engineering. Furthermore, synthetic zinc-finger protein transcription factors can now be designed to target specific pathway enzymes, such as tocopherol methyltransferases. When multiple steps in a pathway are upregulated, previously unsuspected facets of the pathway might be revealed, such as the newly uncovered bifunctional substrate preference of the key regulatory enzyme in tocopherol (vitamin E) biosynthesis, homogentisate phytyltransferase. The engineering of desired traits, such as long-chain omega-3 polyunsaturated fatty acids, can require entirely new pathways to be introduced into a plant. Recent advances in genomics and gene expression technology have made this type of complex metabolic engineering highly feasible.