Intron-mediated enhancement of gene expression in maize ( Zea mays L.) and bluegrass ( Poa pratensis L.)

Vain, Philippe; Finer, Kim R.; Engler, Dean E.; Pratt, Richard; Finer, John J.

doi:10.1007/s002990050060

Cited by 15 publications

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“…It has been demonstrated many times that incorporating introns into transgenes has an enhancing effect on gene expression. This phenomenon was observed predominantly in GC-rich monocot genomes [35], but the mechanisms underlying the enhancement of gene expression are not entirely clear, especially when introducing monocot introns into dicot plants [36]. The great number of ESTs in public databases helped us to assemble at least a partial picture of CKX gene families in Hordeum and Triticum species.…”

Section: Discussionmentioning

confidence: 99%

Cytokinin oxidase/dehydrogenase genes in barley and wheat

Galuszka

Frébortová

Werner

et al. 2004

European Journal of Biochemistry

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The cloning of two novel genes that encode cytokinin oxidase/dehydrogenase (CKX) in barley is described in this work. Transformation of both genes into Arabidopsis and tobacco showed that at least one of the genes codes for a functional enzyme, as its expression caused a cytokinindeficient phenotype in the heterologous host plants. Additional cloning of two gene fragments, and an in silico search in the public expressed sequence tag clone databases, revealed the presence of at least 13 more members of the CKX gene family in barley and wheat. The expression of three selected barley genes was analyzed by RT-PCR and found to be organ-specific with peak expression in mature kernels. One barley CKX (HvCKX2) was characterized in detail after heterologous expression in tobacco. Interestingly, this enzyme shows a pH optimum at 4.5 and a preference for cytokinin ribosides as substrates, which may indicate its vacuolar targeting. Different substrate specificities, and the pH profiles of cytokinin-degrading enzymes extracted from different barley tissues, are also presented.Keywords: cereals; cloning; cytokinin oxidase/dehydrogenase; expression; gene family.Cytokinins were initially viewed as factors promoting cell division and differentiation in plants. Since then, however, cytokinins have been shown to control other developmental events, such as the growth of lateral buds, the release of buds from apical dominance, leaf expansion, the delay of senescence, the promotion of seed germination, and chloroplast formation [1]. Naturally occurring cytokinins are mainly N 6 -substituted adenine derivatives that generally contain an isoprenoid or aromatic side-chain. Recently, considerable progress has been made in elucidating the regulation of cytokinin homeostasis during plant growth and development. New molecular biological techniques have allowed for the identification and characterization of genes encoding important enzymes participating in cytokinin metabolic pathways. Genetically engineered plants that overexpress some of these genes were prepared as a tool to study changes in physiological aspects caused by altered cytokinin levels. Seven genes for isopentenyltransferasescytokinin de novo synthesizing enzymes -were identified in the Arabidopsis genome [2-4]. In addition, three novel genes, encoding cytokinin-specific glycosylation enzymes with different substrate specificities, have been described [5][6][7]. The principle of cytokinin catabolism has been studied for many years. Enzymes capable of degrading cytokinins with unsaturated side-chains have been found in many plant tissues [8], but the details of their features and the mechanism of their action remained unknown for a long time owing to their very low content in plant tissues. The ground-breaking cloning of the cytokinin oxidase maize gene ZmCKX1 [9,10] opened up the possibility for more detailed study of cytokinin degradation, both at the molecular and at the biochemical levels. The recombinant maize enzyme is a glycoprotein containing a covalently bound FAD. The...

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Section: Discussionmentioning

confidence: 99%

Cytokinin oxidase/dehydrogenase genes in barley and wheat

Galuszka

Frébortová

Werner

et al. 2004

European Journal of Biochemistry

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“…Moreover, the frequently observed stimulation of expression caused by introns has been termed IME, which are associated with the intron sequences, splicing machinery and intron position (Clancy and Hannah, 2002;Rose, 2008;Gallegos and Rose, 2015). For example, the first intron of Ubi1, the second and the sixth introns of Adh1, third intron of actin and the fourth intron of RpoT in maize (Mascarenhas et al, 1990;Luehrsen and Walbot, 1991;Vain et al, 1996;Bourdon et al, 2001), can stimulate mRNA accumulation and other introns in these genes cannot enhance gene expression. This is also consistent with our observations that deletion of the second intron but not the first intron from myosin-5 gene exhibits inhibition of myosin-5 expression (Fig.…”

Section: Discussionmentioning

confidence: 99%

Regulatory roles of introns in fungicide sensitivity of Fusarium graminearum

Luo

Jia

et al. 2017

Environmental Microbiology

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Although the roles of introns have been much debated in eukaryotic organisms, none of them have been functionally characterized in Fusarium graminearum. In this study, we characterized the roles of introns in regulation of fungicide-sensitivity of F. graminearum. β tub, cyp51A and myosin-5 are important target genes of benzimidazoles, triazoles and cyanoacrylates respectively. To explore the sensitivity regulation functions of introns in target genes, several detailed deletion studies were completed on the intronic regions of β tub, cyp51A and myosin-5. Phenotypic analyses showed that deletion of the fourth intron from β tub gene (designated β Δi4), the sole intron from cyp51A gene (cyp51A-Δi) and the second intron from myosin-5 gene (myo5-Δi2) exhibited an increased sensitivity to corresponding fungicides. In contrast, deletion of the first or second intron from β tub gene exhibited a decreased sensitivity to carbendazim. qRT-PCR showed that the mRNA transcript levels of target genes were significantly downregulated in β Δi4, cyp51A-Δi and myo5-Δi2 respectively. Meanwhile, Western blot assays revealed that the protein expression levels of β tub was also dramatically reduced in β Δi4, but accumulated in β Δi1 and β Δi2. Overall, our results indicate that introns in target genes significantly regulate the fungicide-sensitivity by influencing expression of the corresponding resident genes in F. graminearum.

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“…From Twyman et al 2003, reproduced with permission of Trends Biotechnol., Vol. 21, p. 573, # 2003 promoter by 100-fold (Vain et al 1996). While the intronmediated enhancement effect may be as high as 100-fold, generally the effect is often 2-to 10-fold, and there tends to be a greater effect in monocots than in dicots, although it is unclear how the enhancing effect is achieved.…”

Section: Enhancement Of Recombinant Protein Accumulation Levels Via Pmentioning

confidence: 95%

“…While the intronmediated enhancement effect may be as high as 100-fold, generally the effect is often 2-to 10-fold, and there tends to be a greater effect in monocots than in dicots, although it is unclear how the enhancing effect is achieved. Some evidence suggests that (i) the presence of introns increases steady-state levels of mRNAs; (ii) although splicing is neither necessary nor sufficient to mediate the intron-enhancing effect, the mRNAs generated by splicing are more rapidly and efficiently exported from the nucleus to the cytoplasm; and (iii) while specific regulatory elements are found within some introns, a more general intron-mediated enhancement is thought to result from synergistic interactions between the factors involved in the various steps of gene expression from transcription to translation (Luehrsen and Walbot 1991;Vain et al 1996;Rose and Beliakoff 2000;Rose 2004 and references therein). Using b-glucuronidase as a reporter, studies by Rose (2004) suggest that there is an intron-mediated enhancement of both transcription and translation.…”

Section: Enhancement Of Recombinant Protein Accumulation Levels Via Pmentioning

confidence: 99%

Plants as factories for production of biopharmaceutical and bioindustrial proteins: lessons from cell biologyThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

Kermode

2006

Can. J. Bot.

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Transgenic plants, seeds, and cultured plant cells are potentially one of the most economical systems for largescale production of recombinant proteins for industrial and pharmaceutical uses. Biochemical, technical, and economic concerns with current production systems have generated enormous interest in developing plants as alternative production systems. However, various challenges must be met before plant systems can fully emerge as suitable, viable alternatives to current animal-based systems for large-scale production of biopharmaceuticals and other products. Aside from regulatory issues and developing efficient methods for downstream processing of recombinant proteins, there are at least two areas of challenge: (1) Can we engineer plant cells to accumulate recombinant proteins to sufficient levels? (2) Can we engineer plant cells to post-translationally modify recombinant proteins so that they are structurally and functionally similar to the native proteins? Attempts to improve the accumulation of a recombinant protein in plant cells require an appreciation of the processes of gene transcription, mRNA stability, processing, and export, and translation initiation and efficiency. Likewise, many post-translational factors must be considered, including protein stability, protein function and activity, and protein targeting. Moreover, we need to understand how the various processes leading from the gene to the functional protein are interdependent and functionally linked. Manipulation of the post-translational processing machinery of plant cells, especially that for N-linked glycosylation and glycan processing, is a challenging and exciting area. The functions of N-glycan heterogeneity and microheterogeneity, especially with respect to protein function, stability, and transport, are poorly understood and this represents an important area of cell biology.Résumé : Les plantes, les graines et les cellules végétales cultivées transgéniques constituent potentiellement un des systè-mes les plus économiques pour la production à grande échelle de protéines recombinantes, pour des usages industriels et pharmaceutiques. Les contraintes biochimiques, techniques et économiques des systèmes de production actuels ont généré un énorme intérêt pour le développement des plantes comme systèmes de production alternatif. Cependant, divers défis doivent être rencontrés, avant que des systèmes végétaux puissent émerger comme alternatives adaptées et viables aux systèmes actuels basés sur les animaux, pour la production à grande échelle de produits biopharmaceutiques et autres. En plus des problèmes de réglementations et du développement de méthodes efficaces pour le traitement en aval des protéines recombinantes, on trouve au moins deux groupes de défis : (1) Pouvons-nous programmer les cellules végétales de manière à accumuler les protéines recombinantes en teneurs suffisamment élevées? (2) Pouvons-nous programmer les cellules végé-tales pour la modification post-traductionnelle des protéines recombinantes de manière à ce qu'elles s...

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Intron-mediated enhancement of gene expression in maize ( Zea mays L.) and bluegrass ( Poa pratensis L.)

Cited by 15 publications

References 0 publications

Cytokinin oxidase/dehydrogenase genes in barley and wheat

Cytokinin oxidase/dehydrogenase genes in barley and wheat

Regulatory roles of introns in fungicide sensitivity of Fusarium graminearum

Plants as factories for production of biopharmaceutical and bioindustrial proteins: lessons from cell biologyThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

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