ISSN 0095-4527, Cytology and Genetics, 2007, Vol. 41, No. 3, pp. 172–175. © Allerton Press, Inc., 2007. Original Russian Text © N.V. Kuchuk, 2007, published in Tsitologiya i Genetika, 2007, Vol. 41, No. 3, pp. 50–54.
Transgenic, Transplastomic, and Transient Approaches to Alien Gene Expression in Plants N. V. Kuchuk Institute of Cell Biology and Genetic Engineering, National Academy of Sciences, ul. Zabolotnogo 150, Kyiv, 031443 Ukraine e-mail:
[email protected] Received December 15, 2006
Abstract—Recent experiments are reviewed, which aimed at developing the transgenic and transplastomic plants, as well as at accumulating recombinant proteins due to transient expression. These experiments were performed in the Department of Genetic Engineering of the Institute of Cell Biology and Genetic Engineering of the National Academy of Science, Ukraine. The new approaches have been developed to induce promoterless gene expression in transgenic plants and to obtain transplastomic plants with the use of “clipboard” species. DOI: 10.3103/S0095452707030061
Extended studies on the genetic transformation of plants are carried out in the Department of Genetic Engineering of the Institute of Cell Biology and Genetic Engineering of the National Academy of Science, Ukraine. Development of the method of genetic transformation has a significant place and transgenic plants of some agricultural crops have been already obtained. These are transgenic plants of sugar beet [1], rape [2], tomato (V. Rudas), potato (E. Kishchenko), pea [3], and haricot (S. Nifantova) (table). The methods of genetic transformation have their own particular features in the
breeding each of these species and domestically produced genotypes, varieties, and lines are primarily used. Along with the selective genes, some genes responsible for resistance to herbicides were helpful in genetic transformation. We have developed rape and sugar beet transgenic plants that are resistant to BASTA, as well as pea and haricot plants resistant to Pivot herbicide. Along with the conventional techniques of gene transfer, alternative methods are now studied to induce alien gene transfer and expression. Transposon activity is widely used for mapping and cloning of plant genes.
Transgenic plants developed in the Department of Genetic Engineering, Institute of Cell Biology and Genetic Engineering Plant species Sugar beet (Beta vulgaris) The same "
Rape (Brassica napus) The same Tomato (Lycopersicon esculentum) Potato (Salanum tuberosum) Tobacco Aftican (Nicotiana Africana) Pea (Pisum sativum) Pea (Pisum sativum) Hericot (Phaseolus vulgaris)
Selective trait
The studied or agriculturally valuable traits
Resistance to kanamycin Activity of Spm/dSpm transposon system Resistance to phosphinothricin Resistance to BASTA herbicide Resistance to phosphinothricin and kanamycin The effect of lox site on expression of the promoterless bar gene Resistance to kanamycin Resistance to Pivot herbicide Resistance to phosphinothricin Resistance to BASTA herbicide Resistance to phosphinothricin and kanamycin The effect of lox site on expression of the promoterless bar gene Resistance to kanamycin Expression of the reporter gene Resistance to phosphinothricin and kanamycin The effect of lox site on expression of the promoterless bar gene Resistance to phosphinothricin and kanamycin The effect of lox site on expression of the promoterless bar gene Resistance to phosphinothricin Resistance to BASTA herbicide Resistance to kanamycin Resistance to Pivot herbicide Resistance to kanamycin Resistance to Pivot herbicide 172
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Insertion of a heterologous transposon system into the genome of plants that are of practical importance, though they lack their own similar system, may be helpful in cloning of unique genes. In addition, a functional transposon system makes it possible to readily eliminate undesired genetic markers that get into the genome together with the genetic construction used. The transposon system may facilitate transfer of the desired gene from the genome of the transformed line to that of the selective lines in the course of subsequent sexual crossings. The lack of genetic linkage in the case of gene transposition facilitates transfer of the desired trait into different varieties of the same species under sexual hybridization between the transgenic form and selective lines. In our experiments with selective sugar beet lines, several transgenic plants were obtained which carried the Spm maize system (E. Kishchenko). Transferring interspecific transposons from one genome to another in remote sexual or somatic hybrids is an interesting scientific task that along with its fundamental significance could have also a practical outcome. Gene transfer between the genomes of different species due to activity of maize Spm transposons was studied by the methods of somatic hybridization. Somatic hybrids of mustard or rape and Arabidopsis were obtained [4–6]. Wild-type mustard or rape plants were used, i.e., they did not have specific genetic traits within the range of the variation we studied. Arabidopsis was previously transformed using a construction where the gene responsible for resistance to the herbicide BASTA was flanked with dSpm sequences and could be transposed due to the activity of a specific transposase whose gene was integrated into the same construction. Transposition was detected using GUS gene activity that occurs only in case of excision of the transposed element together with the gene bar that separates the GUS gene structural region and promoter in the original vector construction. In remote somatic hybrids, such as intertribal Brassica + Arabidopsis, elimination of one of the genomes was expected. The condition chosen promoted elimination of the Arabidopsis genome, whereas the mustard or rape genome should be retained. The regenerating colonies were selected by resistance to BASTA herbicide. In these experiments, plants resistant to BASTA, which however resembled rape or mustard, were selected. These plants were flowering, but generated no progeny at this point. Analysis of isoenzymes and ITSrepeat variation did not reveal the Arabidopsis genome in these plants. However, the transposase and GUS genes that are closely associated with the Arabidopsis genome were always detectable among transgenes along with the bar gene that could be transposed into the rape or mustard genome. Thus, transposable gene transfer between the genomes of the unstable hybrid combinations of mustard + Arabidopsis and rape + Arabidopsis was not clearly confirmed because certain transgene-associated regions of the Arabidopsis genome seem to be retained in our hybrids. NevertheCYTOLOGY AND GENETICS
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less, these results provide a somewhat different view on the possibility of long-term retention of the genes of a species whose genome is eliminated from remote unstable hybrids. Similar results were obtained with the sexual hybrids of Nicotiana tabacum x Nicotiana Africana. The data on the expression of genes that lack their own promoter in the original genetic constructions is of interest. In this study, the genetic elements from Crelox specific recombination system were used. The promoterless bar gene responsible for resistance to either ammonium glucosinate or to BASTA herbicide was integrated in different positions into the T-DNA of the Ti plasmid from Agrobacterium tumefaciens and an additional element, the lox site, was either present or absent before the encoding gene regions. When the promoterless gene occurs near the right T-DNA border, it is known to be expressed occasionally in transgenic plants because a plant gene may be integrated under a promoter. However, the frequency of even this expression is extremely low, reaching, at best, 20% of all the selected transformants. We also obtained a few herbicide-resistant plants (about 1%) when the promoterless bar gene was in this position. However, if the lox site occurred between the structural gene region and the right border of the T-DNA, the number of resistant plants increased strongly to reach 80% of the plants selected initially for resistance to another selective marker—kanamycin. The gene responsible for resistance to kanamycin was also found in the genetic constructions used, and the transformants were first selected for this trait. At the same time, when the promoterless gene was far from the right border, even together with the lox site, the researchers failed to observe the above phenomenon [2, 7, 8]. We obtained similar results in a series of plants belonging to different families (sugar beet, tobacco, potato, and rape). Thus, promoterless genes may be used for development of genetically modified plants in the case where the alien DNA is delivered with the use of agrobacteria. “Direct” genetic transformation of tobacco with the same constructions, by means of bombing, failed to induce the above phenomenon, suggesting that the agrobacterial mechanisms participate in the integration and expression of the promoterless genes. Among our experiments, the development of transplantomic plants, i.e., those carrying alien genetic information within the plastome rather than in nuclear genome, plays an important role. Transplantome plants of the economically valuable species promise some advantages over the nuclear transformants. These are the high level of expression of the transferred gene, the possibility of polycistronic regulation, and ecological safety due to the absence of the alien gene in the pollen. However, all these advantages are overridden by the large problems associated with gene transfer into the chloroplast genome. Actually, tobacco is the only subject in which this transformation has been reproducible in different laboratories. We have developed a new
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method of chloroplast transformation with the use of an intermediate, so-called “clipboard,” plant [9–11]. In this method, plastids of the plant species of interest are transferred by means of somatic hybridization into the plants of a species that can be readily cultivated and regenerated in vitro. After transformation of the chloroplast DNA and development of a transplastomic plant cybrid, chloroplansts were again transferred into the cells of the original parent. Using this technology, the transformed plastomes were obtained in different species of the nightshade family [12–17]. We have also developed rape transplastomic plants carrying transformed chloroplasts from Lesquerella fendleri [18]: Transformed plastome
Plant species carrying the transformed plastome
Atropa belladonna Scopolia carniolica Physochlaina officinalis Lycium barbarum Lycium barbarum Salpiglossis sinuata Salpiglossis sinuata Solanum rickii Solanum rickii Lesquerella fendleri
Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Lycium barbarum Nicotiana tabacum Salpiglossis sinuata Solanum rickii Lycopersicon peruvianum Brassica napus
In respect to current plant biotechnology, these are not only the subjects of agrarian production, but also biological systems capable of producing pharmaceutical proteins and vaccines in the same manner as microorganisms and animal cells. Moreover, plant biological systems are closer to animals and man than microorganisms, and therefore proteins that cannot be produced by bacteria could be synthesized in plants. This production requires expenses that are dozens of times lower than the same production in mammalian cells. In respect to biosafety, these plants are also more advantageous than animal cells and bacteria, because no viruses, prions, or endotoxins have been identified in plants that could be potentially dangerous for humans. In the Department of Genetic Engineering, a rather simple system has been developed, where, after injection of a special gene combination into the leaves of several Australian tobacco species, the plant’s proteinsynthesizing system is completely switched over to the synthesis of the desired product [19]. As early as a week after such an injection, the amount of the desired protein in a plant leaf reaches 5–80% of the total protein contents of this plant. This approach may revolutionize the current pharmacological industry such that greenhouses will be used instead of either fermenters with a bacterial suspension or the very expensive systems that maintain mammalian cell growth. Thus, we have not only studied new pathways of transfer and expression of genetic information in
plants. Possibilities have been created for the development in Ukraine of plants that can be successfully used for farming to improve productivity via protection from biotic and abiotic stresses, as well as for production of pharmaceutical proteins and vaccines. REFERENCES 1. Kishchenko, O.M., Komarnytskii, I.K., Gleba, Yu.Yu., and Kuchuk, M.V., Development of Transgenic Plants of Sugar Beet Beta vulgaris, O-Type Lines, Using Agrobacterium tumefaciens, Tsitologiya Genetika, 2004, vol. 38, no. 5, pp. 3–8. 2. Sakhno, L.A., Getsko, I.O., Komarnytskii, I.K., and Kuchuk, N.V., The Plants Brassica napus and Orychophragmus violaceus (Brassicaceae) Transformed with Cre-lox-containing constructions, Faktory of experimental’noi evolutsii organisma (The Factors of Experimental Organisms Evolution): Poika M. V. ed.; Kiev: Agrar. Nauka, 2003, pp. 366–371. 3. Nifantova, C.N., Simonenko, Yu.V., Komarnytskii, I.K., and Kuchuk, N.V., Development of Transgenic Sowing Pea Plants (Pisum sativum L.) Resistant to the Herbicide Pursuit, Tsitologiya Genetika, 2005, vol. 39, no. 6, pp. 16–21. 4. Sachno, L.A., Sytnik, E.S., Cherep, N.N., Komarnytskii, I.K., Kuchuk, N.V., and Klimyuk, V.I., Activity of Maize Spm Transposon System in Transgenic Plants Orychophragmus violaceus L. O. E. Shultz, Which Were Obtained Using Both Direct DNA Transferring into Protoplasts and Agrobacterial Transformation of Root Explants, Tsitologiya Genetika, 2002, vol. 36, no. 6, pp. 3–8. 5. Ovcharenko, O.O., Komarnytskii, I.K., Cherep, M.M., Gleba, Yu.Yu., Kuchuk, M.V., Development and Analysis of Somatic Brassica napus + Arabidopsis thaliana, Hybrids Containing Heterologous System of Maize Spm/dSpm Transplosones, Tsitologiya Genetika, 2005, vol. 39, no. 3, pp. 50–56. 6. Ovcharenko, O.O., Komarnytskii, I.K., Cherep, M.M., Rudas, V.A., Kuchuk, M.V., Development of Intertribe Somatic Digenomic (Orychophragmus voilaceus + Arabidopsis Thaliana) and Tetragenomic (Orychophragmus violaceus + Brassica juncea + Arabidopsis Thaliana) Hybrids and the Use for Studying the Heterologous Spm/dSpm Transposon System, Biopolymery i Kletka, 2005, vol. 21, no. 1, pp. 35–41. 7. Shcherbak, N.L., Belokurova, V.B., Komarnytskii, I.K., and Kuchuk, N.V., Genetic Transformation of Plants Nicatiana africana Merxm. by Plasmids Containing lox Recombination Site, Tsytolog. Genetika, 2004, vol. 38, no. 4, pp. 3–8. 8. Shcherbak, N.L., Belokurova, V.B., Getsko, I.O., Komarnytskii, I.K., and Kuchuk, N.V., The Effect of the lox Site of Cre-lox Recombination System on Expression of the Promoterless bar Gene in Transgenic Plants, Tsytol. Genetika, 2006, vol. 40, no. 1, pp. 3–9. 9. Vasilenko, M.Yu., Komarnytskii, I.K., Sakhno, L.A., Gleba, Yu.Yu., and Kuchuk, N. V., Development and Analysis of Intergeneral Somatic Hybrids of Brassica napus and albino-Type Line of Orychaphragmus violaceus, Tsitol. Genetika, 2003, vol. 37, no. 1, pp. 3–10. CYTOLOGY AND GENETICS
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