pEmu: an improved promoter for gene expression in cereal cells

Summary A recombinant promoter, pEmu, has been constructed to give a high level of gene expression in monocots. It is based on a truncated maize Adh1 promoter, with multiple copies of the Anaerobic Responsive Element from the maize Adh1 gene and ocs-elements from the octopine synthase gene of Agrobacterium tumefaciens. The pEmu promoter was one of 12 different promoter constructs that were linked to the -glucuronidase (GUS) marker gene. Promoter activity was measured 48 h after introduction of the constructs into protoplasts of five different monocot species [wheat, maize, rice, einkorn (Triticum monococcum), and Lolium multiflorum] and one dicot (Nicotiana plumbaginifolia). In suspension cell protoplasts, the most highly expressing construct (pEmuGN) gave 10- to 50-fold higher expression than the CaMV 35S promoter in all the monocot species. The pEmu promoter should be valuable where a high level of gene expression is required in monocots. The pEmu promoter showed instability in several widely used Escherichia coli strains but was stable in a recA, recD strain AC001, which is described. Another construct, p4OCS35SIGN, gave a tenfold increase in expression over the CaMV 35S promoter in dicot (Nicotiana plumbaginifolia) protoplasts.     

Comparative analysis of rice MADS-box genes expressed during flower development

MADS-box genes involved in flower development have been isolated and studied in a wide variety of plant species. However, most of these studies are related to dicot species like Antirrhinum majus, Arabidopsis thaliana and Petunia hybrida. Although the floral structures of typical monocot and dicot flowers differ substantially, previous studies indicate that MADS-box genes controlling floral organ identity in dicots can also be identified in monocot plants like rice and maize. To extend this study further to obtain a more global picture of monocot and dicot MADS-box gene evolution, we performed a phylogenetic study using MADS-box genes from A. thaliana and Oryza sativa. Furthermore, we investigated whether the identified orthologues of Arabidopsis and rice have a conserved expression profile that could indicate conservation of function.     

A promoter from sugarcane bacilliform badnavirus drives transgene expression in banana and other monocot and dicot plants

A 1369 bp DNA fragment (Sc) was isolated from a full-length clone of sugarcane bacilliform badnavirus (ScBV) and was shown to have promoter activity in transient expression assays using monocot (banana, maize, millet and sorghum) and dicot plant species (tobacco, sunflower, canola and Nicotiana benthamiana). This promoter was also tested for stable expression in transgenic banana and tobacco plants. These experiments showed that this promoter could drive high-level expression of the -glucuronidase (GUS) reporter gene in most plant cells. The expression level was comparable to the maize ubiquitin promoter in standardised transient assays in maize. In transgenic banana plants the expression levels were variable for different transgenic lines but was generally comparable with the activities of both the maize ubiquitin promoter and the enhanced cauliflower mosaic virus (CaMV) 35S promoter. The Sc promoter appears to express in a near-constitutive manner in transgenic banana and tobacco plants. The promoter from sugarcane bacilliform virus represents a useful tool for the high-level expression of foreign genes in both monocot and dicot transgenic plants that could be used similarly to the CaMV 35S or maize polyubiquitin promoter.     

Production of fertile transgenic barley (Hordeum vulgare L.) plant using the hygromycin-resistance marker

Fertile transgenic barley (Hordeum vulgare L.) plants were obtained by high velocity particle bombardment. The plasmid pBCl was used to deliver the selectable hph gene and reporter Gus gene into immature embryo. After the selection culture 18 hygromycin resistant plants were obtained. Samples for Southern hybridization and enzymatic Gus assay were obtained from 11 plants. Southern hybridization confirmed the presence of the hph gene in the 11 hygromycin resistant plants(T₀). Enzymatic assay indicated that all the t₀ plants that showed hph positive in Southern analysis possessed detectable amount of Gus activity. To date all the 11 t₀ plants reached maturity and mature seeds were obtained Transmission of the hph gene to progeny(T₁) of two independent t₀ plants was confirmed by Southern hybridization.Abbreviations Adh Alcohol Dehydrogenase - BA 6-Benzylaminopurine - cv cultivar - 2,4-D 2,4-Dichlorophenoxyacetic Acid - Gus -Glucuronidase - hph Hygromycin Phosphotransferase - 4MU 4-Methyl-umbelliferone     

Ntlim1, a PAL-box binding factor,controls promoter activity of the horseradish wound-inducible peroxidase gene

To understand molecular mechanisms underlying wound-induced expression of plant peroxidase genes, the promoter of a horseradish C2 peroxidase (prxC2) gene was analyzed. We had previously isolated a tobacco nuclear protein, Ntlim1, as a trans factor binding to a PAL-box motif of the prxC2 promoter; however, the function of the Ntlim1 trans factor and the PAL-box motif in wound-responsive expression of the prxC2 gene remains unclear. Here, we found that the prxC2 promoter without the intact PAL-box motif failed to direct a normal level of both the basal and the wound-induced expression of -glucuronidase (GUS) reporter gene in transgenic tobacco plants, indicating that the PAL-box motif functions as an essential cis element of the prxC2 promoter. We also found that antisense expression of Ntlim1 in transgenic plants carrying the prxC2 promoter::GUS chimeric construct decreased not only the level of the basal and the wound-induced expression of the GUSreporter gene but also the extent of wound inducibility of the prxC2 promoter itself. This result indicates that Ntlim1 is required for the basal level of prxC2 promoter activity as well as its up-regulation under wound stress. Moreover, consistent with the results obtained in planta, result from super-shift assay indicates that the Ntlim1 binds to the PAL-box motif independently of wound stress.     

利用烟草瞬时表达系统检测高等植物基因的剪接加工

解析基因的剪接加工机制是了解植物形态建成、生长发育和逆境胁迫应答的重要环节．与动物相比,植物中相应的研究进展较为缓慢．利用农杆菌介导的烟草瞬时表达系统,分别对单子叶植物水稻BADH2和双子叶植物拟南芥GR7基因片段在烟草叶片中的转录后剪接加工进行分析．结果表明,一些重要剪接调控元件在植物中保守存在,而烟草瞬时表达系统可以作为研究高等植物剪接调控的重要工具,快捷灵敏地检测基因的剪接加工方式．     

Co-expression of a Modified Maize Ribosome-inactivating Protein and a Rice Basic Chitinase Gene in Transgenic Rice Plants Confers Enhanced Resistance to Sheath Blight

Chitinases, -1,3-glucanases, and ribosome-inactivating proteins are reported to have antifungal activity in plants. With the aim of producing fungus-resistant transgenic plants, we co-expressed a modified maize ribosome-inactivating protein gene, MOD1, and a rice basic chitinase gene, RCH10, in transgenic rice plants. A construct containing MOD1 and RCH10 under the control of the rice rbcS and Act1 promoters, respectively, was co-transformed with a plasmid containing the herbicide-resistance gene bar as a selection marker into rice by particle bombardment. Several transformants analyzed by genomic Southern-blot hybridization demonstrated integration of multiple copies of the foreign gene into rice chromosomes. Immunoblot experiments showed that MOD1 formed approximately 0.5% of the total soluble protein in transgenic leaves. RCH10 expression was examined using the native polyacrylamide-overlay gel method, and high RCH10 activity was observed in leaf tissues where endogenous RCH10 is not expressed. R₁ plants were analyzed in a similar way, and the Southern-blot patterns and levels of transgene expression remained the same as in the parental line. Analysis of the response of R₂ plants to three fungal pathogens of rice, Rhizoctonia solani, Bipolaris oryzae, and Magnaporthe grisea, indicated statistically significant symptom reduction only in the case of R. solani (sheath blight). The increased resistance co-segregated with herbicide tolerance, reflecting a correlation between the resistance phenotype and transgene expression.     

Development of transgenic sorghum for insect resistance against the spotted stem borer (Chilo partellus)

Transgenic sorghum plants expressing a synthetic cry1Ac gene from Bacillus thuringiensis (Bt) under the control of a wound-inducible promoter from the maize protease inhibitor gene (mpiC1) were produced via particle bombardment of shoot apices. Plants were regenerated from the transformed shoot apices via direct somatic embryogenesis with an intermittent three-step selection strategy using the herbicide Basta. Molecular characterisation based on polymerase chain reaction and Southern blot analysis revealed multiple insertions of the cry1Ac gene in five plants from three independent transformation events. Inheritance and expression of the Bt gene was confirmed in T₁ plants. Enzyme-linked immunosorbant assay indicated that Cry1Ac protein accumulated at levels of 1–8 ng per gram of fresh tissue in leaves that were mechanically wounded. Transgenic sorghum plants were evaluated for resistance against the spotted stem borer (Chilo partellus Swinhoe) in insect bioassays, which indicated partial resistance to damage by the neonate larvae of the spotted stem borer. Reduction in leaf damage 5 days after infestation was up to 60%; larval mortality was 40%, with the surviving larvae showing a 36% reduction in weight over those fed on control plants. Despite the low levels of expression of Bt -endotoxin under the control of the wound-inducible promoter, the transgenic plants showed partial tolerance against first instar larvae of the spotted stem borer.     

The rice metallothionein gene promoter does not direct foreign gene expression in seed endosperm

We generated transgenic tobacco and rice plants harboring a chimeric gene consisting of the 5-upstream sequence of the rice metallothionein gene (ricMT) fused to the -glucuronidase (GUS) gene. The activity and tissue-specific expression of the ricMT promoter were demonstrated in these transgenic plants. In the transgenic rice plants, despite substantial levels of GUS activity in the shoot and root, almost no GUS signal was detected in the endosperm. Thus, the ricMT promoter could be useful in avoiding accumulation of undesired proteins in the seed endosperm.     

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

We report a strength comparison of a large variety of monocot and dicot intron-containing fragments inserted in the 5 untranslated leader, between the CaMV 35S promoter and the uidA gene (coding for the ß-glucuronidase: GUS). Relative strengths of the intron-containing fragments were evaluated by comparing transient GUS expression after particle bombardment in embryogenic maize and bluegrass suspension cultures. Our results confirm a dramatic dependence on the presence of an intron for chimeric gene expression in both species. On average, the maize first intron of ubi1 provided the highest enhancement of gene expression in maize and bluegrass (71- and 26-fold enhancement, respectively). Half of the introns tested affected gene expression differently in bluegrass and maize. This suggests that the intron-mediated enhancement of gene expression generally obtained with maize may not be fully applicable to all monocots. We also report enhancement of gene expression (92-fold) in a monocot species by a dicot intron (chsA intron).     

Characterization of Transgenic Rice Plants Expressing an Arabidopsis FAD7

Fatty acid -3 desaturase (FAD) is the key enzyme catalyzing the formation of trienoic fatty acids. We utilized an Arabidopsis FAD7 gene and the seven independent transgenic rice plants harbouring 1 to 3 copies of this gene were generated. The expression of FAD7 mRNA was different among independent transgenic lines regardless of the copy number. The total linolenic acid (18:3) contents reduced by about 7 – 32 % in transgenic rice plants but the linoleic acid (18:2) content increased accordingly. With or without wounding treatments, the jasmonate content was higher in transgenic lines than in wild-type rice plant. The transgenic lines overproducing jasmonate also showed increased expression of PR1b mRNA and allene oxide synthase inresponse to wounding.     

Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice

Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot‐and‐mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound‐ and pathogen‐inducible mpi promoter. The mpi‐pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi‐pci rice, compared with larvae fed on wild‐type plants, was observed. Expression of the mpi‐pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi‐pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi‐pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi‐pci fusion gene for dual resistance against insects and pathogens in rice plants.     

Cloning and Characterization of AKR4C14, a Rice Aldo–Keto Reductase, from Thai Jasmine Rice

Aldo–keto reductase (AKR) is an enzyme superfamily whose members are involved in the metabolism of aldehydes/ketones. The AKR4 subfamily C (AKR4C) is a group of aldo–keto reductases that are found in plants. Some AKR4C(s) in dicot plants are capable of metabolizing reactive aldehydes whereas, such activities have not been reported for AKR4C(s) from monocot species. In this study, we have screened Indica rice genome for genes with significant homology to dicot AKR4C(s) and identified a cluster of putative AKR4C(s) located on the Indica rice chromosome I. The genes including OsI_04426, OsI_04428 and OsI_04429 were successfully cloned and sequenced by qRT-PCR from leaves of Thai Jasmine rice (KDML105). OsI_04428, later named AKR4C14, was chosen for further studies because it shares highest homology to the dicot AKR4C(s). The bacterially expressed recombinant protein of AKR4C14 was successfully produced as a MBP fusion protein and his-tagged protein. The recombinant AKR4C14 were capable of metabolizing sugars and reactive aldehydes i.e. methylglyoxal, a toxic by-product of the glycolysis pathway, glutaraldehyde, and trans-2-hexenal, a natural reactive 2-alkenal. AKR4C14 was highly expressed in green tissues, i.e. leaf sheets and stems, whereas flowers and roots had a significantly lower level of expression. These findings indicated that monocot AKR4C(s) can metabolize reactive aldehydes like the dicot AKR4C(s) and possibly play a role in detoxification mechanism of reactive aldehydes.