GUS Reporter Gene Research Articles

Molecular cloning and analysis of a receptor-like promoter of Gbvdr3 gene in sea island cotton.

Verticillium wilt caused by soil borne fungus Verticillium dahliae could significantly reduce cotton yield. The Ve1 homologous gene Gbvdr3 is resistant to Verticillium wilt. In order to understand of the function of the promoter Gbvdr3 in Gossypium barbadense, the promoter region of the receptor-like gene Gbvdr3 was obtained by genome walking, and the cis-element in the promoter was identified using the PLACE software in this study. The sequence analysis showed that the promoter contained elements related to stress resistance and light regulation. The cloned promoter was fused to the GUS reporter gene and transformed into Arabidopsis. GUS expression was specifically detected in roots, flowers, and seeds, suggesting that the expression of Gbvdr3 is tissue-specific. Separation and characterization analysis of the promoter of Gbvdr3 provides a platform for further research and application of this gene. Thorough understanding of the function of the Gbvdr3 promoter is important for better understanding of Gbvdr3 function. These results indicated that the promoter of Gbvdr3 was a tissue-specific promoter.

Efficient seed-specifically regulated autoexcision of marker gene (nptII) with inducible expression of interest gene in transgenic Nicotiana tabacum

In this study, we developed the seed-specifically regulated autoexcision system based on Cre/loxP site-specific recombination for coelimination of nptII and cre genes from transgenic plants. To accomplish this, a seed-specific gene promoter, BcNA1, from Brassica napus was used to drive conditional expression of recombinase. NptII and recombinase cassettes were placed between two directly repeated loxP sites. The loxP-flanked DNA was located between the SP-DDEE synthetic pathogen inducible promoter and pr??omoterless β-glucuronidase (Gus) gene, as a model gene of interest. Upon seed-specific expression of cre recombinase, the excision event would eliminate loxP-flanked DNA and bring the gus reporter gene under the control of the inducible promoter. This Cre/loxP system was transformed into Nicotiana tabacum via Agrobacterium-??mediated transformation. The regenerated plants were obtained by selection on kanamycin medium and PCR screening. Based on seed germination assay on kanamycin-containing medium, the transgenic lines were subdivided into homogeneous and heterogeneous categories. Phenotypic and molecular analysis of T1 progeny plants indicated that completely NptII-free plants were obtained in homogeneous transgenic lines. Coelimination of NptII and recombinase cassettes were verified with PCR, sequencing, and histochemical Gus staining assay. Full excision efficiency (100%) demonstrates the effectiveness of this autoexcision system for the production of marker gene-free transgenic plants.

Establishment of Agrobacterium-Mediated Genetic Transformation in Popular Rice Cultivar BRRI Dhan-29 through Reporter GUS Gene Expression System in Bangladesh

Establishment of Agrobacterium-Mediated Genetic Transformation in Popular Rice Cultivar BRRI Dhan-29 through Reporter GUS Gene Expression System in Bangladesh

A High-Throughput Regeneration and Transformation Platform for Production of Genetically Modified Banana.

Banana (Musa spp.) is an important staple food as well as cash crop in tropical and subtropical countries. Various bacterial, fungal, and viral diseases and pests such as nematodes are major constraints in its production and are currently destabilizing the banana production in sub-Saharan Africa. Genetic engineering is a complementary option used for incorporating useful traits in banana to bypass the long generation time, polyploidy, and sterility of most of the cultivated varieties. A robust transformation protocol for farmer preferred varieties is crucial for banana genomics and improvement. A robust and reproducible system for genetic transformation of banana using embryogenic cell suspensions (ECS) has been developed in this study. Two different types of explants (immature male flowers and multiple buds) were tested for their ability to develop ECS in several varieties of banana locally grown in Africa. ECS of banana varieties “Cavendish Williams” and “Gros Michel” were developed using multiple buds, whereas ECS of “Sukali Ndiizi” was developed using immature male flowers. Regeneration efficiency of ECS was about 20,000–50,000 plantlets per ml of settled cell volume (SCV) depending on variety. ECS of three different varieties were transformed through Agrobacterium-mediated transformation using gusA reporter gene and 20–70 independent transgenic events per ml SCV of ECS were regenerated on selective medium. The presence and integration of gusA gene in transgenic plants was confirmed by PCR, dot blot, and Southern blot analysis and expression by histochemical GUS assays. The robust transformation platform was successfully used to generate hundreds of transgenic lines with disease resistance. Such a platform will facilitate the transfer of technologies to national agricultural research systems (NARS) in Africa.

English

The plasmids pULGU1 and pEmuGN were introduced by biolistics in embryogenic cell suspensions of pearl millet, Pennisetum glaucum . The plasmid pULGU1 contained the mutant acetolactate synthase (ALS) gene of Arabidopsis thaliana , responsible for resistance to the chlorsulfuron herbicide. The plasmid pEmuGN carried the reporter β-glucuronidase (GUS) gene of Escherichia coli . Two months after bombardment of the cells, transformed calli were selected on Murashige and Skoog medium containing 30 nM chlorsulfruron, a completely concentration inhibitory for non-transformed cells. A concentration of 200 nM chlorsufuron did not affect the growth of these selected resistant calli. Genomic DNA of resistant cells presented an electrophoretic pattern revealing the integration of the mutant ALS gene into the genome of Pennisetum glaucum . Expression of the GUS reporter gene was revealed by histochemical assay of the blue coloration of the calli in the presence of the substrate Xgluc (5-bromo-4-chloro-3-indolyl-β-D-Glucuronic acid). The presence of GUS gene was furthermore confirmed, by southern blot hybridization of non-radioactive probes, labelled with digoxigenin, on genomic DNA extracted from selected chlorsulfuron-resistant calli. GUS gene activity observed in all selected calli was high during the first 6 months after bombardment and then decreased. Pearl millet plants were regenerated from cell lines derived from chlorsulfuron resistant calli. Southern blot hybridization of non-radioactive probes with genomic DNA extracted from these regenerated plants showed the presence of the GUS and ALS transgenes, confirming the stable transformation of these plants. Key words : Pennisetum glaucum , stable transformation, chlorsulfuron (ALS), β-glucuronidase (GUS), CaMV35S and Emu promoters.

Characterization of the Populus PtrCesA4 promoter in transgenic Populus alba × P. glandulosa

An efficient shoot regeneration method was developed using leaf explants of Populus alba × P. glandulosa, and the optimized shoot regeneration medium contained 0.1 mg L−1 NAA, 0.5 mg L−1 6-BA and 0.002 mg L−1 TDZ. The factors for Agrobacterium-mediated transformation of this hybrid were further optimized as follows: no preculture step, Agrobacterium cell suspension with an OD600 of 0.6, 20 min infection time and 2d co-cultivation duration. To investigate Populus trichocarpa cellulose synthase A4 promoter (PtrCesA4pro), we constructed the PtrCesA4pro:: β-glucuronidase (GUS) binary vector, in which the cauliflower mosaic virus 35S (CaMV35S) promoter of pBI121was replaced with the PtrCesA4pro to drive the expression of a GUS reporter gene. Forty-eight kanamycin-resistant plantlets were obtained through Agrobacterium-mediated transformation of this hybrid. Genomic DNA PCR and Southern blot analyses confirmed the integration of PtrCesA4pro::GUS into this hybrid and the expression of GUS gene was driven by the PtrCesA4 promoter. A histochemical GUS staining presented the activity of PtrCesA4 promoter in the transgenic lines. No GUS signal was observed in leaves, including the veins and petioles, and notably weak staining was detected in the secondary xylems of the roots. The PtrCesA4 promoter was highly active in the fibers, the vessels of developing xylems, and the fibers of mature phloems. Surprisingly, GUS staining was detected in the cambial cells of the highly lignified stems of these transgenic trees. Our results implicate PtrCesA4 promoter as a good genetic tool for controlling gene function in wood development and tree molecular breeding.

Vascular expression of Populus LRR-RLK genes and the effects of their overexpression on wood formation

Leucine-rich repeat receptor-like kinases (LRR-RLKs) constitute the largest subfamily of receptor-like kinases (RLKs) in land plants. Although some LRR-RLKs have been characterized as playing key roles in cell-to-cell communications, the physiological roles of most LRR-RLKs remain to be determined. In this study, we first screened the Populus trichocarpa expressed sequence tag and microarray database to look for PtLRR-RLKs differentially and preferentially expressed across the vascular cambial zone. The cell-specific expression of seven selected PtLRR-RLK genes was then verified by the GUS (β-glucuronidase) reporter gene assay. Nearly all of the seven types of plants transformed with PtLRR-RLKpro:GUS fusions showed a high level of transcription in the developing primary xylem and interfascicular cambium during the early stages of xylem differentiation. In stems undergoing secondary growth, the GUS activities driven by the seven PtLRR-RLK promoters were mainly associated with the cambial zone. These seven PtLRR-RLK genes were then individually overexpressed in hybrid poplars, and most transgenic poplars did not exhibit significant phenotypic changes. However, overexpression of PtLRR-RLK1, the putative ortholog of TDR/PXY in Arabidopsis, led to an ectopic accumulation of lignin in the pith along with an enlarged secondary xylem and increased lignin content without perceptible effect on secondary cell wall thickness, which made this transgene a likely candidate as a transducer of signals during plant secondary growth and wood formation.

Efficient heat-shock removal of the selectable marker gene in genetically modified grapevine

Cisgenesis is one of the new plant breeding technologies emerging as a promising tool for the future, more publicly accepted than the traditional transgenic approach. One of the requirements for a cisgenic plant is the absence of selectable marker genes in the genome. In this study, a system for marker gene removal after selection of transgenic plants has been tested in grapevine. This is based on a binary vector containing a heat-shock-inducible promoter which, upon induction, activates a recombinase to produce the excision of a FRT-flanked box. After the removal of this cassette hosting both the marker gene, nptII, and the recombinase itself, the reporter gene gus may be expressed. Gene transfer experiments on grapevine embryogenic callus were carried out via Agrobacterium tumefaciens. Different heat-shock treatments with variable temperatures and heat incubations times were tested on a selected line and the optimal conditions for a complete removal of nptII with the subsequent gus transcription were found. Plants were analysed by means of qPCR on genomic DNA, to quantify nptII removal, and by a fluorimetric assay to measure gus activity. Our study is conceived as a proof-of-concept to investigate the feasibility of this method in grapevine in view of developing an efficient cisgenic approach in this valuable fruit crop.

Fusarium Oxysporum Volatiles Enhance Plant Growth Via Affecting Auxin Transport and Signaling.

Volatile organic compounds (VOCs) have well-documented roles in plant-plant communication and directing animal behavior. In this study, we examine the less understood roles of VOCs in plant-fungal relationships. Phylogenetically and ecologically diverse strains of Fusarium oxysporum, a fungal species complex that often resides in the rhizosphere of assorted plants, produce volatile compounds that augment shoot and root growth of Arabidopsis thaliana and tobacco. Growth responses of A. thaliana hormone signaling mutants and expression patterns of a GUS reporter gene under the auxin-responsive DR5 promoter supported the involvement of auxin signaling in F. oxysporum volatile-mediated growth enhancement. In addition, 1-naphthylthalamic acid, an inhibitor of auxin efflux, negated F. oxysporum volatile-mediated growth enhancement in both plants. Comparison of the profiles of volatile compounds produced by F. oxysporum strains that differentially affected plant growth suggests that the relative compositions of both growth inhibitory and stimulatory compounds may determine the degree of plant growth enhancement. Volatile-mediated signaling between fungi and plants may represent a potentially conserved, yet mostly overlooked, mechanism underpinning plant-fungus interactions and fungal niche adaption.

Characterization of 5'UTR of rice ClpB-C/Hsp100 gene: evidence of its involvement in post-transcriptional regulation.

Rice (Oryza sativa) ClpB-C (OsClpB-C) protein is expressed upon heat stress in vegetative tissues and constitutively in seeds. We produced stably transformed Arabidopsis plants carrying β-glucuronidase (Gus) reporter gene downstream to 1-kb OsClpB-C promoter (1kbPro plants). In the 1kbPro plants, expression of Gus transcript and protein followed the expression pattern of OsClpB-C gene in rice plants, i.e., heat induced in vegetative tissues and constitutive in seeds. Next, we produced transgenic Arabidopsis plants containing Gus downstream to 862-bp fragment of OsClpB-C promoter [lacking 138 nucleotides from 3' end of the 5'untranslated region (5'UTR); ∆UTR plants). In ∆UTR plants, Gus transcript was expressed in heat-inducible manner, but strikingly, Gus protein levels were negligible after heat treatment. However, Gus protein was expressed in ∆UTR seedlings at levels comparable to 1kbPro seedlings when recovery treatment of 22 °C/2 h was given post heat stress (38 °C/15 min). This suggests that 5'UTR of OsClpB-C gene is involved in its post-transcriptional regulation and is an obligate requirement for protein expression during persistent heat stress. Furthermore, the Gus transcript levels were higher in the polysomal RNA fraction in heat-stressed seedlings of 1kbPro plants as compared to ∆UTR plants, indicating that 5'UTR aids in assembly of ribosomes onto the Gus transcript during heat stress. Unlike the case of seedlings, Gus protein was formed constitutively in ∆UTR seeds at levels comparable to 1kbPro seeds. Hence, the function of 5'UTR of OsClpB-C is dispensable for expression in seeds.

Molecular study on the carAB operon reveals that carB gene is required for swimming and biofilm formation in Xanthomonas citri subsp. citri.

BackgroundThe carA and carB genes code the small and large subunits of carbamoyl-phosphate synthase (CPS) that responsible for arginine and pyrimidine production. The purpose of this work was to study the gene organization and expression pattern of carAB operon, and the biological functions of carA and carB genes in Xanthomonas citri subsp. citri. MethodsRT-PCR method was employed to identify the full length of carAB operon transcript in X. citri subsp. citri. The promoter of carAB operon was predicted and analyzed its activity by fusing a GUS reporter gene. The swimming motility was tested on 0.25 % agar NY plates with 1 % glucose. Biofilm was measured by cell adhesion to polyvinyl chloride 96-well plate.ResultsThe results indicated that carAB operon was composed of five gene members carA-orf-carB-greA-rpfE. A single promoter was predicted from the nucleotide sequence upstream of carAB operon, and its sensitivity to glutamic acid, uracil and arginine was confirmed by fusing a GUS reporter gene. Deletion mutagenesis of carB gene resulted in reduced abilities in swimming on soft solid media and in forming biofilm on polystyrene microtiter plates.ConclusionsFrom these results, we concluded that carAB operon was involved in multiple biological processes in X. citri subsp. citri.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0555-9) contains supplementary material, which is available to authorized users.

Production of transgenic apricot plants from hypocotyl segments of mature seeds

Apricot (Prunus armeniaca L.) is one of the most recalcitrant species for genetic transformation. In this work; we report the regeneration of transgenic apricot plants from mature seed hypocotyl segments (cv. ‘Canino’ and ‘Moniquí’) for the first time. Moreover, this is the first report of the regeneration of apricot transgenic plants by means of the selective bar gene. Agrobacterium tumefaciens strain LB4404 harbouring a binary plasmid with eyfp and nptII genes (pMOG-eyfp-IV2) or a plasmid with gus and bar genes (pFGC5941-gus), was used in this study. A gradual selection strategy with aminoglycoside antibiotics made it possible to produce transgenic plants with 1.5±0.5% efficiency using 10 μM paromomycin (PAR). When buds were isolated and placed in a meristem development medium, PAR was changed to 20μM kanamycin (KAN). As the buds developed and elongated, the concentration was increased up to 140μM KAN based on a 20μM increase every 4 weeks. When bar was the marker gene, the regeneration-inhibitory PPT concentration (75.7μM) was applied immediately after the co-culture. In this case, the PPT concentration was reduced afterwards to 10.1μM in the micropropagation medium. This strategy allowed for the regeneration of transgenic shoots with 3.8±1.4% efficiency. Transformation events were monitored by detection of the reporter gene (eyfp or gus) expression. PCR and Southern blot analysis confirmed the stable integration of the transgenes into the apricot genome. Transformed shoots were rooted on a selective medium, acclimatised, cultured and raised under greenhouse conditions. Our results confirm that the process used in this study is an efficient transformation system for the apricot species which could be used for functional genomics studies and the development of transgenic rootstocks.

The Elucidation of the Interactome of 16 Arabidopsis bZIP Factors Reveals Three Independent Functional Networks.

The function of the bZIP transcription factors is strictly dependent on their ability to dimerize. Heterodimerization has proven to be highly specific and is postulated to operate as a combinatorial mechanism allowing the generation of a large variety of dimers with unique qualities by specifically combining a small set of monomers; an assumption that has not yet been tested systematically. Here, the interaction pattern and the transactivation properties of 16 Arabidopsis thaliana bZIPs are examined in transiently transformed Arabidopsis protoplasts to deliver a perspective on the relationship between bZIP dimerization and function. An interaction matrix of bZIPs belonging to the C, G, H, and S1 bZIP groups was resolved by Bimolecular Fluorescent Complementation (BiFC) coupled to quantitative flow cytometric analysis, while an extensive GUS reporter gene assay was carried out to determine the effect of different bZIP pairs on the expression of four different known bZIP-targeted promoters. Statistical data treatment and complementary bioinformatic analysis were performed to substantiate the biological findings. According to these results, the 16 bZIPs interact in three isolated networks, within which their members dimerize non-specifically and exhibit a significant level of functional redundancy. A coherent explanation for these results is supported by in silico analysis of differences in the length, structure and composition of their leucine zippers and appears to explain their dimerization specificity and dynamics observed in vivo quite well. A model in which the bZIP networks act as functional units is proposed.

Characterization and Transcriptional Profiling of Ginkgo biloba Mevalonate Diphosphate Decarboxylase Gene (GbMVD) Promoter Towards Light and Exogenous Hormone Treatments

Mevalonate diphosphate decarboxylase (GbMVD) of Ginkgo biloba catalyzes the final step of mevalonic acid pathway by converting the six-carbon mevalonate diphosphate into isopentenyl diphosphate, a precursor of ginkgolides and bilobalide collectively termed terpene trilactones (TTLs). This paper presents the isolation of a 1.2-kb fragment (designated as GbMVDpro) of the 5′-flanking region of GbMVD. Extensive sequence analysis revealed the presence of evolutionarily conserved putative cis-acting elements in light-regulated transcription, hormone signaling (abscisic acid, jasmonate, and salicylic acid), and plant defense (W-box/WRKY) in the GbMVD promoter region. Electrophoretic mobility shift analysis suggested possible involvement of W-box in GbMVD promoter function. To assess the organ-specific and environmentally responsive characteristics of GbMVD expression, GbMVDpro was fused to GUS reporter gene. GbMVDpro::GUS was introduced into tobacco. Histological analysis of the transgenic tobacco showed that GUS mainly localized in the leaf epidermis, stem secondary xylem, and root vasculature. These observations closely correlated with the previously reported presence of GbMVD transcripts in the roots, stems, and leaves. Further functional characterizations in transiently transformed tobacco leaves allowed us to identify the region that can be considered as the minimal promoter. Light and hormonal signals (i.e., ethephon, salicylic acid, and methyl jasmonate) induced the activity of GbMVDpro-driven GUS in transgenic tobacco and enhanced the transcription of GbMVD as well as TTL production in ginkgo seedlings. These results, together with the bioinformatic analysis of GbMVDpro, suggest that the GbMVD promoter harbored a numerous TTL biosynthesis-related cis-elements that regulate the flux of terpenoid precursors.

Functional Characterization of NAC and MYB Transcription Factors Involved in Regulation of Biomass Production in Switchgrass (Panicum virgatum).

Switchgrass is a promising biofuel feedstock due to its high biomass production and low agronomic input requirements. Because the bulk of switchgrass biomass used for biofuel production is lignocellulosic secondary walls, studies on secondary wall biosynthesis and its transcriptional regulation are imperative for designing strategies for genetic improvement of biomass production in switchgrass. Here, we report the identification and functional characterization of a group of switchgrass transcription factors, including several NACs (PvSWNs) and a MYB (PvMYB46A), for their involvement in regulating secondary wall biosynthesis. PvSWNs and PvMYB46A were found to be highly expressed in stems and their expression was closely associated with sclerenchyma cells. Overexpression of PvSWNs and PvMYB46A in Arabidopsis was shown to result in activation of the biosynthetic genes for cellulose, xylan and lignin and ectopic deposition of secondary walls in normally parenchymatous cells. Transactivation and complementation studies demonstrated that PvSWNs were able to activate the SNBE-driven GUS reporter gene and effectively rescue the secondary wall defects in the Arabidopsis snd1 nst1 double mutant, indicating that they are functional orthologs of Arabidopsis SWNs. Furthermore, we showed that PvMYB46A could activate the SMRE-driven GUS reporter gene and complement the Arabidopsis myb46 myb83 double mutant, suggesting that it is a functional ortholog of Arabidopsis MYB46/MYB83. Together, these results indicate that PvSWNs and PvMYB46A are transcriptional switches involved in regulating secondary wall biosynthesis, which provides molecular tools for genetic manipulation of biomass production in switchgrass.

The upstream regulatory sequence of the light harvesting complex Lhcf2 gene of the marine diatom Phaeodactylum tricornutum enhances transcription in an orientation- and distance-independent fashion

Diatoms are a key phytoplankton group in the contemporary ocean, showing extraordinary adaptation capacities to rapidly changing environments. The recent availability of whole genome sequences from representative species has revealed distinct features in their genomes, like novel combinations of genes encoding distinct metabolisms and a significant number of diatom-specific genes. However, the regulatory mechanisms driving diatom gene expression are still largely uncharacterized. Considering the wide variety of fields of study orbiting diatoms, ranging from ecology, evolutionary biology to biotechnology, it is thus essential to increase our understanding of fundamental gene regulatory processes such as transcriptional regulation. To this aim, we explored the functional properties of the 5'-flanking region of the Phaeodatylum tricornutum Lhcf2 gene, encoding a member of the Light Harvesting Complex superfamily and we showed that this region enhances transcription of a GUS reporter gene in an orientation- and distance-independent fashion. This represents the first example of a cis-regulatory sequence with enhancer-like features discovered in diatoms and it is instrumental for the generation of novel genetic tools and diatom exploitation in different areas of study.

Developing fiber specific promoter-reporter transgenic lines to study the effect of abiotic stresses on fiber development in cotton.

Cotton is one of the most important cash crops in US agricultural industry. Environmental stresses, such as drought, high temperature and combination of both, not only reduce the overall growth of cotton plants, but also greatly decrease cotton lint yield and fiber quality. The impact of environmental stresses on fiber development is poorly understood due to technical difficulties associated with the study of developing fiber tissues and lack of genetic materials to study fiber development. To address this important question and provide the need for scientific community, we have generated transgenic cotton lines harboring cotton fiber specific promoter (CFSP)-reporter constructs from six cotton fiber specific genes (Expansin, E6, Rac13, CelA1, LTP, and Fb late), representing genes that are expressed at different stages of fiber development. Individual CFSP::GUS or CFSP::GFP construct was introduced into Coker 312 via Agrobacterium mediated transformation. Transgenic cotton lines were evaluated phenotypically and screened for the presence of selectable marker, reporter gene expression, and insertion numbers. Quantitative analysis showed that the patterns of GUS reporter gene activity during fiber development in transgenic cotton lines were similar to those of the native genes. Greenhouse drought and heat stress study showed a correlation between the decrease in promoter activities and decrease in fiber length, increase in micronaire and changes in other fiber quality traits in transgenic lines grown under stressed condition. These newly developed materials provide new molecular tools for studying the effects of abiotic stresses on fiber development and may be used in study of cotton fiber development genes and eventually in the genetic manipulation of fiber quality.

KiwiPME1 encoding pectin methylesterase is specifically expressed in the pollen of a dioecious plant species, kiwifruit (Actinidia chinensis)

Pectin methylesterases (PMEs) mediate demethylesterification of pectic polysaccharides such as homogalacturonan, a major component of the primary plant cell wall, in the apoplasm. The PMEs are implicated in a number of developmental processes, including pollen development and pollen tube growth, through the fine tuning of the methylesterification status of pectin. In this study, we isolated a full-length cDNA (KiwiPME1) encoding PME from kiwifruit and characterized its molecular features. Analyses of the primary protein structure and gene structure revealed that KiwiPME1 encodes a pre-pro-PME protein that is predicted to localize to the outside of the cell and belongs to group 2 (formerly type 1). The KiwiPME1 expression was highly detected in pollen grains of kiwifruit but not in vegetative tissues investigated. Expression pattern analysis of KiwiPME1 among different floral tissues of male and female plants revealed that KiwiPME1 was expressed specifically in the stamens of flower buds in male and female plants, whereas its expression was detected only in the stamens of male plants when the flowers opened. Expression analysis of KiwiPME1 promoter fused to the GUS reporter gene in Arabidopsis displayed a very similar pattern to that in kiwifruit. Our study suggests that the cell wall-localized KiwiPME1 is likely implicated in the pollen development and pollen tube growth of a dioecious species kiwifruit.

Functional Analysis of the Maize C-Repeat/DRE Motif-Binding Transcription Factor CBF3 Promoter in Response to Abiotic Stress.

The ZmCBF3 gene is a member of AP2/ERF transcription factor family, which is a large family of plant-specific transcription factors that share a well-conserved DNA-binding domain. To understand the regulatory mechanism of ZmCBF3 gene expression, we isolated and characterized the ZmCBF3 promoter (PZmCBF3). Three deletion fragments of PZmCBF3 were generated, C1–C3, from the translation start codon at position −1079, −638, and −234, and fused to the GUS reporter gene. Each deletion construct was analyzed by Agrobacterium-mediated stable transformation and expression in Arabidopsis thaliana. GUS expression assays indicated that the PZmCBF3 exhibited root-specific expression activity. A 234-bp fragment upstream of the ZmCBF3 gene conferred a high level of GUS activity in Arabidopsis. Some cis-acting elements involved in the down-regulation of gene expression were detected in the promoter, encompassing positions −1079 to −234. PZmCBF3 was activated by cold stress. The MYCCONSENSUSAT elements (CANNTG) were responsible for the ability of PZmCBF3 to respond to cold stress. The results of the present study suggest that PZmCBF3 might play a role in cold tolerance in maize.

Truncated Ubiquitin 5' Regulatory Region from Erianthus arundinaceus Drives Enhanced Transgene Expression in Heterologous Systems.

Characterization of novel plant gene promoters underpins the development of transgenic crop plants. Here, we report a novel 5' regulatory sequence (Eriubi D7) of the ubiquitin gene from Erianthus arundinaceus, a wild relative of sugarcane resistant to many biotic and abiotic stresses. A 3.2-kb regulatory sequence of ubiquitin gene was isolated through random amplification of genomic ends technique and characterized in rice, tobacco, and sugarcane. In silico analysis revealed that the regulatory sequence contained a promoter region of 1600bp upstream to the transcription start site. Between the promoter and the coding region, two putative introns of 584 and 583bp and two putative non-coding exons of 459 and 37bp were spaced alternatively. To identify the active domains required for gene regulation, 12 truncations/recombinants were made in the regulatory sequence and characterized in heterologous systems. Transformation studies with the recombinant constructs revealed that Eriubi D7, a truncated fragment containing 830bp promoter and the intron I, conferred enhanced GUS reporter gene expression in both monocots and dicots compared to other routinely used promoters such as maize ubi1 and Cauliflower Mosaic Virus 35S. Further analysis confirms that this regulatory sequence is quite distinct from the other reported ubiquitin promoters and was also found to enhance expression of the reporter gene upon wounding. This is the first report on the isolation and characterization of a promoter from a wild sugarcane germplasm and is expected to be useful for development of transgenic crop plants.