GUS Reporter Gene Research Articles

An auxin responsive CLE gene regulates shoot apical meristem development in Arabidopsis.

Plant hormone auxin regulates most, if not all aspects of plant growth and development, including lateral root formation, organ pattering, apical dominance, and tropisms. Peptide hormones are peptides with hormone activities. Some of the functions of peptide hormones in regulating plant growth and development are similar to that of auxin, however, the relationship between auxin and peptide hormones remains largely unknown. Here we report the identification of OsCLE48, a rice (Oryza sativa) CLE (CLAVATA3/ENDOSPERM SURROUNDING REGION) gene, as an auxin response gene, and the functional characterization of OsCLE48 in Arabidopsis and rice. OsCLE48 encodes a CLE peptide hormone that is similar to Arabidopsis CLEs. RT-PCR analysis showed that OsCLE48 was induced by exogenously application of IAA (indole-3-acetic acid), a naturally occurred auxin. Expression of integrated OsCLE48p:GUS reporter gene in transgenic Arabidopsis plants was also induced by exogenously IAA treatment. These results indicate that OsCLE48 is an auxin responsive gene. Histochemical staining showed that GUS activity was detected in all the tissue and organs of the OsCLE48p:GUS transgenic Arabidopsis plants. Expression of OsCLE48 under the control of the 35S promoter in Arabidopsis inhibited shoot apical meristem development. Expression of OsCLE48 under the control of the CLV3 native regulatory elements almost completely complemented clv3-2 mutant phenotypes, suggesting that OsCLE48 is functionally similar to CLV3. On the other hand, expression of OsCLE48 under the control of the 35S promoter in Arabidopsis has little, if any effects on root apical meristem development, and transgenic rice plants overexpressing OsCLE48 are morphologically indistinguishable from wild type plants, suggesting that the functions of some CLE peptides may not be fully conserved in Arabidopsis and rice. Taken together, our results showed that OsCLE48 is an auxin responsive peptide hormone gene, and it regulates shoot apical meristem development when expressed in Arabidopsis.

A library of synthetic transcription activator-like effector-activated promoters for coordinated orthogonal geneexpression in plants.

A library of synthetic promoters containing the binding site of a single designer transcription activator-like effector (dTALE) was constructed. The promoters contain a constant sequence, consisting of an 18-base long dTALE-binding site and a TATA box, flanked by degenerate sequences of 49 bases downstream and 19 bases upstream. Forty-three of these promoters were sequenced and tested in transient assays in Nicotiana benthamiana using a GUS reporter gene. The strength of expression of the promoters ranged from around 5% to almost 100% of the viral 35S promoter activity. We then demonstrated the utility of these promoters for metabolic engineering by transiently expressing three genes for the production of a plant diterpenoid in N. benthamiana. The simplicity of the promoter structure shows great promise for the development of genetic circuits, with wide potential applications in plant synthetic biology and metabolic engineering.

Agrobacterium tumefaciens mediated genetic transformation of Ocimum gratissimum: A medicinally important crop

Ocimum gratissimum is renowned for its immense role in traditional, ayurvedic and unani system of medicines. The essential oil has a plethora of active ingredient like triterpenes, polyphenols and phenylpropanoids. The active ingredients claim most of the properties like anticancerous, anti HIV, antipyretic, antinociceptive, antiurolithiatic etc. Screening of biologically active ingredients from this valuable plant may provide us the basis to produce novel pharmaceutically important drugs and medicines. Considering the importance of this plant, it is the need to have a well developed regeneration and transformation protocol.The present report focuses on the development of an efficient protocol for direct regeneration and Agrobacterium mediated genetic transformation of O. gratissimum. CN; when inoculated on MS+1.5mg/L+0.5mg/L+0.5mg/L KN medium has shown the best response with a regeneration frequency of 98%±0.4. A. tumefaciens LBA4404 containing binary vector pBI121, harboring gus-A reporter gene with intron under the transcriptional control of Cauliflower Mosaic Virus (CaMV) 35S promoter and neomycin phosphotransferase (npt-II) as selection marker genes, to achieve maximum transformation frequency (20%±0.7). To carry out transformation, parameters like O.D., infection duration, concentration of AS, surfactant, (O.D600-0.6, 15min, 300μM, 0.01%) were standardized. The putative transformants were screened on SMM having 50mg/L kanamycin and subsequently rooted on ½ MS. The confirmation was done through PCR. The established regeneration and transformation protocol could be used as a tool to elucidate the functional genomics of important/novel compound which can be used in the preparation of therapeutic formulations for future.

Plant Growth Promotion Assessment in Rice Plant Enhanced by Inoculation of Rhizobacteria

In the search of efficient biofertilizer, nine efficient strains of PGPR were evaluated by inoculation in two different rice varieties, Saryu-52 and Malviya Dhan-36 in the gnotobiotic conditions using two different media FCN-medium and soil extract medium. Agrobacterium sp. strain BN-2A showed better result in respect to other eight isolates in the total length, fresh weight, number of roots and chlorophyll-a content. By the inoculation Agrobacterium sp. strain BN-2A, total length increased in Saryu-52 (22.6%) and in Malviya Dhan-36 (52.1%), fresh weight increased in Saryu-52 (42.4%) and in Malviya Dhan-36 (68.8%) and chlorophyll-a increased in Saryu-52 (76.6%) and in Malviya Dhan-36 (37.1%). Similarly in soil extract medium, inoculation of Agrobacterium sp. strain BN-2A alone showed better result in comparison to mixture of nine strains. To prove that colonization indeed occurs, gusA reporter gene was tagged with the most efficient isolate Agrobacterium sp. strain BN-2A and colonization in the rice root was confirmed by gusA staining and histochemical analysis of gusA staining. Therefore, BN-2A has best potential to be used as biofertilizer.DOI: http://dx.doi.org/10.3126/av.v4i0.12362Academic Voices Vol.4 2014: 73-84

Transcriptional profiling of genes encoding GABA-transaminases in Brassica napus reveals their regulation by water deficit

In plants, GABA (γ-aminobutyric acid) accumulates in response to a wide range of environmental stresses. The metabolism of this non-protein amino acid occurs in two distinct cellular compartments: it is synthesized in the cytosol and degraded in mitochondria. Although many studies have reported its involvement in development, the role of stress-induced GABA accumulation remains unclear. The effects of GABA accumulation have been examined in plants defective in GABA catabolism. In Arabidopsis mutants deficient for GABA transaminase (GABA-T), the first step in GABA degradation, it was shown to be involved in salt stress tolerance. Here, we investigated GABA-T genes in the Brassicaceae crop species Brassica napus at the molecular structure and transcriptional levels. We show that several copies of GABA-T are ubiquitously expressed in many organs of oilseed rape. Analysis of BnaGABA-T promoters using the GUS reporter gene found no difference in localization between GABA-T family members. Additionally, we show that their expression profiles are modified during development and in response to water stress depending on leaf rank. The role of GABA-T regulation in water stressed B. napus plants is discussed.

Characterization and Functional Analysis of Transcription Factor LoMYB80 Related to Anther Development in Lily (Lilium Oriental Hybrids)

Anther development is very precise and complicated procedure. In this study, the gene LoMYB80 was isolated from lily (Lilium Oriental Hybrids cv. cultivar ‘Siberia’) anther. Multiple alignment and phylogenetic tree analysis showed that LoMYB80 belongs to the R2R3 MYB family. Furthermore, the protein of LoMYB80 shares a 44 amino acid sequence immediately adjacent to the R3 domain with other MYB80s. Subcellular localization showed that LoMYB80 was predominantly localized in the nucleus. The transcriptional activation experiment revealed that LoMYB80 belongs to the transcription activation factors and its transactivation activity depends on the last 70 amino acids located at the end of the C-terminal region. Real-time quantitative PCR analysis showed that LoMYB80 gene expression was significantly higher in anther than in other organs; the highest level was in anthers of 10-cm flower buds and then was in anthers of 4-cm buds. The relative expression in microspores/pollen of 10-cm buds was much higher than the whole anther in the same stage and also much higher than the microspore/pollen of 8- and 9-cm buds. The gene was mainly expressed in young anthers at the development stages from 6 to 10 by analyzing transgenic Arabidopsis lines which were transformed with LoMYB80 promoter::GUS reporter gene constructs. Complementation experiments revealed that transgenic ms188 mutant lines could produce normal pollen grains and display partial fertility. These results indicated that LoMYB80 was a regulator of lily anther development and pollen formation.

Cloning and Transient Expression Analysis of Promoter of PnsICE1 from Populus simonii×P.nigra

ICE1 encodes a MYC-like b HLH transcriptional activator,which could specially bind to active domain of CBF3 promoter and induce transcriptal expression of cold-responsive genes downstream of CBF3,play important roles in stress response. A 1 247 bp sequence of Pns ICE1 promoter was cloned from DNA of Populus simonii × P. nigra by PCR amplification method. By sequence analysis,it consisted of a typical core promoter region of eukaryotic,beside the basic elements: TATA-box and CAAT-box,and there were some hormone responsive elements and multiple stress-induced elements of ABRE,DOFCOREZM,MYBCORE,MYCCONSENSUSATHSE and W-box. Pns ICE1 promoter may associated with adversity stress and play important roles in response to various stresses in P. simonii × P. nigra. To verify the biology function,recombinant vector was designated as p BI121-ICEPro through replacing Ca MV35 S promoter in p BI121 by cloned Pns ICE1 promoter fragment. In the vector,gus reporter gene was driven by Pns ICE1 promoter. GUS histochemical assay in Arabidopsis thaliana after transient infection showed that gus expressed in both flower and root.

Agrobacterium-mediated Genetic Transformation in Cucumber (var. Shital) as Influenced by Explant, Inoculation Time and Co-cultivation Period

To study genetic transformation for abiotic stress resistance in cucumber (var. Shital), leaf, nodal and internodal calli were subjected to Agrobacterium tumefaciens mediated transformation using LBA4404 strain containing CIPK sense gene. Transformation ability was examined by histochemical assay of GUS reporter gene in survived calli. Conspicuous GUS positive (blue colour) region were detected in callus tissue. There were 3 factors in this investigation. Factor A consisted of three types of explants viz. leaf, nodal and internodal callus, factor B consisted of two durations of inoculation time viz. 3 and 5 min and factor C consisted of two co-cultivation periods viz. 24 and 48 hours. The highest GUS positive transgenic callus obtained from leaf explants (3.30) and internodal explants gave the lowest number (1.79) of GUS positive transformants. Inoculation time is an important factor in transformation experiment mediated by A. tumefaciens. Transformation ability was increased with increase of inoculation time. Percentage of survived callus was higher (53.68 %) when the calli were immersed for 5 min in bacterial suspension. Both number and percentage of GUS positive callus were higher (3.17 and 52.87 %, respectively) when they were kept in bacterial suspension for higher time (5 min) and lower (2.15 and 35.88 %, respectively) when calli were soaked in bacterial suspension for minimum time (3 min).

Using leaf explants for transformation of Chrysanthemum morifolium ramat. mediated by Agrobacterium tumefaciens

An Agrobacterium-mediated transformation for chrysanthemum using leaf explants of two different cultivars was conducted. Leaves were first infected with Agrobacterium tumefaciens LBA4404 strain, harbouring a binary vector pBI121 carrying the GUS reporter gene and the nptII as the selectable marker gene. 30 seconds for infection of leaves proved to be the optimum duration. Best results were achieved when explants were cocultivated with A. tumefaciens for two days. Shoot induction medium was supplemented with 7 mgl -1 kanamycin and 300 mgl -1 cefotaxime. Putative transgenic plants were obtained after rooting on 1/2 MS medium containing 7mgl-1 kanamycin in the absence of cefotaxime. Transformation efficiency of ‘Reagan Elite Salmon’ (29.1%) was higher than ‘Resomee Splendid’ (9.1%). Presence of the gus gene was verified with histochemical GUS assay and polymerase chain reaction (PCR) of transformed plants.

Differential regulation of genes by retrotransposons in rice promoters.

Rice genome harbors genes and promoters with retrotransposon insertions. There is very little information about their function. The effect of retrotransposon insertions in four rice promoter regions on gene regulation, was investigated using promoter-reporter gene constructs with and without retrotransposons. Differences in expression levels of gus and egfp reporter genes in forward orientation and rfp in reverse orientation were evaluated in rice plants with transient expression employing quantitative RT-PCR analysis, histochemical GUS staining, and eGFP and RFP fluorescent microscopy. The presence of SINE in the promoter 1 (P1) resulted in higher expression levels of the reporter genes, whereas the presence of LINE in P2 or gypsy LTR retrotransposon in P3 reduced expression of the reporter genes. Furthermore, the SINE in P1 acts as an enhancer in contrast with the LINE in P2 and the gypsy LTR retrotransposon in P3 which act as silencers. CTAA and CGG motifs in these retrotransposons are the likely candidates for the downregulation compared to TCTT motif (SINE) which is a candidate for the upregulation of gene expression. The effect of retrotransposons on gene regulation correlated with the earlier investigation of conservation patterns of these four retrotransposon insertions in several rice accessions implying their evolutionary significance.

Isolation and molecular characterization of a novel WIN1/SHN1 ethylene-responsive transcription factor TdSHN1 from durum wheat (Triticum turgidum. L. subsp. durum).

Over the last decade, APETALA2/Ethylene Responsive Factor (AP2/ERF) proteins have become the subject of intensive research activity due to their involvement in a variety of biological processes. This research led to the identification of AP2/ERF genes in many species; however, little is known about these genes in durum wheat, one of the most important cereal crops in the world. In this study, a new member of the AP2/ERF transcription factor family, designated TdSHN1, was isolated from durum wheat using thermal asymetric interlaced PCR (TAIL-PCR) method. Protein sequence analysis showed that TdSHN1 contained an AP2/ERF domain of 63 amino acids and a putative nuclear localization signal (NLS). Phylogenetic analysis showed that TdSHN1 belongs to a group Va protein in the ERF subfamily which contains the Arabidopsis ERF proteins (SHN1, SHN2, and SHN3). Expression of TdSHN1 was strongly induced by salt, drought, abscisic acid (ABA), and cold. In planta, TdSHN1 protein was able to activate the transcription of GUS reporter gene driven by the GCC box and DRE element sequences. In addition, TdSHN1 was targeted to the nucleus when transiently expressed in tobacco epidermal cells. In transgenic yeast, overexpression of TdSHN1 increased tolerance to multiple abiotic stresses. Taken together, the results showed that TdSHN1 encodes an abiotic stress-inducible, transcription factor which confers abiotic stress tolerance in yeast. TdSHN1 is therefore a promising candidate for improvement of biotic and abiotic stress tolerance in wheat as well as other crops.

Character of oligosaccharin OS-RG participation in the IAA-induced formation of adventitious roots

The interaction between auxin and oligosaccharin OS-RG of natural origin (DP ∼10) during the process of adventitious root formation was studied. Oligosaccharin isolated from pea seedlings increased by 20–25% the number of roots induced by IAA on both the segments of buckwheat (Fagopyrum esculentum Moench) hypocotyls and explants produced from the leaves of transgenic (rolB-GUS) tobacco Nicotianatabacum L., cv Petit Havana. The highest effect was obtained after short-term treatment of explants with oligosaccharin before hormone adding. The optimal time of pretreatment depended on the used model system and was from 1–2 to 5–24 h for buckwheat hypocotyl segments and explants from tobacco leaves, respectively. Treatment with OS-RG after IAA did not affect the number of roots induced by the hormone. By using the explants from the leaves of transgenic tobacco plants harboring the reporter GUS gene under the control of the auxin-inducible promoter of the rolB gene permitted to reveal the explant response to the hormone at the early stages of root formation. The dynamics of GUS activity after IAA addition was characterized by the presence of two peaks. The histological analysis showed that the first peak coincided with the formation of 4–5-layer primordia, whereas the second peak — with the emergence of essentially developed roots. Pretreatment of explants from tobacco leaves with OS-RG activated IAA-induced GUS-activity and accelerated the response, i.e., the shift of the first peak to the beginning of culture without change in the position of the second peak. Thus, obtained data indicate that OS-RG action precedes that of the hormone at early stages of rhizogenesis. Possible mechanisms of interaction between IAA and oligosaccharin in the process of root formation are discussed.

Isolation and Characterization of a OsRap2.4A Transcription Factor and its Expression in Arabidopsis for Enhancing High Salt and Drought Tolerance

Dehydration responsive element/C-repeat (DRE/CRT) is a cis-acting element involved in the regulation of abiotic stress-responsive gene expression in higher plants. Using a 50-nucleotide bait containing DRE cis-acting element localized on the downstream mini TATA box of glutamate dehydrogenase-like protein ( JRC2606 ) promoter in yeast one-hybrid screening, we have identified two transcription factors belonging to A6 subgroup of DREB subfamily - OsRap2.4A and OsRap2.4B. Expression of OsRap2.4A was induced by drought, high salt and temperature stress conditions, and also by abscisic acid treatment. Binding assay showed that OsRap2.4A bound specifically to DRE sequence in both in vivo and in vitro experiments. Transient expression of OsRap2.4A in protoplasts revealed that OsRap2.4A functioned as a transcriptional activator and upregulated expression of the GUS re-porter gene. Transgenic Arabidopsis plants expressing OsRap2.4A at low level showed no significant growth re-tardation under normal condition, whereas at high level expression of OsRap2.4A caused an obvious retardation. Expression of OsRap2.4A showed the tolerance against drought and salt stresses in comparison with control. These results suggested that OsRap2.4A gene may be involved in a new regulatory pathway in plant responses to abiotic stresses and it is potentially useful for the transformation into crop plants to improve tolerance against drought and high salt stresses.

Identification and analysis of the mechanism underlying heat-inducible expression of rice aconitase 1

Respiratory metabolism is an important though poorly understood facet of plant adaptation to stress. Posttranslational modification of aconitase, a component of the tricarboxylic acid cycle (TCA), may be involved in stress tolerance. However, such stress-related transcriptional regulation and its mechanism remain unknown. In this study, we found that expression of the rice Aconitase gene OsACO1 is induced in a time-dependent manner by heat but not other typical abiotic stresses. To analyze the transcriptional regulation mechanism underlying the response to heat, the OsACO1 promoter (POsACO1) was isolated and characterized in transgenic rice. Using qualitative and quantitative analyses, we found that the expression of the GUS reporter gene responded to heat in different tissues and at different stages of development when driven by POsACO1. A series of 5′ distal deletions of POsACO1 was generated to delineate the region responsible for heat-induced gene expression. Transient expression analyses in tobacco leaves identified a 322-bp minimal region between −1386 and −1065 as being essential and sufficient for heat-induced expression by POsACO1. We screened for known heat response-related cis-elements in this 322-bp region; however, sequences correlating with heat-induced gene expression were not identified in POsACO1. Therefore, truncations and successive mutagenesis analyses were performed in this 322-bp region. By comparing the activities of promoter fragments and their derivatives, our results indicated that the heat response element resided in a 9-bp region between −1132 and −1124, a sequence that contains a W-box motif. Additional site-directed mutagenesis analyses eliminated the heat response activity of POsACO1 via the W-box element, and an electrophoretic mobility shift assay (EMSA) indicated the binding of POsACO1 by factors in the nuclear extracts of heat-stressed rice seedlings in a W-box-dependent manner. Our results illustrate the expression pattern of a key component of the TCA response to abiotic stress and establish a putative regulatory pathway in the transcriptional modulation of rice respiratory metabolism genes in response to heat.

Regulation of the JUB1 stress-related transcription factor in Arabidopsis thaliana in response to biotrophic fungus Oidium neolycopersici

Biotic stresses influence fitness of the plants and may decrease their productivity. Especially biotrophic fungi govern complex regulation of host metabolism to fulfill their nutritional needs, while the plant tries to avoid this fungal activity. The defense system of the plant may involve transcription of stress-dependent genes, activation of signaling pathways or production of antimicrobial compounds. The responsible signal molecule in biotrophic pathogen-host interaction was found to be mostly the salicylic acid (SA). We studied here, whether the salicylic acid (SA) is required for the induction of a stress-related transcription factor JUB1 by the biotrophic fungus of tomato Oidium neolycopersici or not. To prove the regulation of JUB1, we isolated the upstream region of the gene and transcriptionally fused to gus reporter gene. This promoter::reporter fusion was then transferred into wild type and two salicylic acid insensitive mutants of Arabidopsis thaliana (nim1-1 and nahG). The transgenic plants were infected by powdery mildew (PM) O. neolycopersici. We found an obvious increase of reporter gene expression in all three lines mostly at the area, where the pathogen was contacted to the plants. Based on the result, the stress-dependent JUB1 transcription factor is probably not influenced by the SA mutations of nim1-1 and nahG. However, the induction appeared along with hydrogen-peroxide development, which suggests that this gene regulation in response to powdery mildew is probably regulated via H2O2 and not by SA signaling.

Functional characterization and analysis of the Arabidopsis UGT71C5 promoter region.

In the present study, we isolated an Arabidopsis promoter, UGT71C5, and analyzed its role in the regulation of the light response mechanism. We constructed a fusion vector pBI121-pU-GUS by integrating the UGT71C5 promoter upstream of the GUS reporter gene in pBI121, and then transferred this vector into Arabidopsis plants. The GUS activity of the transgenic plants was detected using a spectrophotometer under normal growth conditions as well as under light, drought, and ABA stress-treatments. The obtained results indicated that the GUS activity of transgenic plants ranged in between the activities observed in wild-type and 35S transgenic plants, which were used as positive control. Light stress for 8 and 12 h increased the GUS activity in transgenic plants by 3 and 4 times, respectively, compared to the activity in these plants under normal conditions. No such change in the GUS activity was observed under drought and ABA-treated conditions. This suggests that the UGT71C5 promoter is light inducible. Our study provides helpful insights into the elucidation of inducible promoters in Arabidopsis and the molecular mechanisms of light response.

Counter-transcribed RNAs of Rhizobium leguminosarum repABC plasmids exert incompatibility effects only when highly expressed

The six plasmids of Rhizobium leguminosarum VF39SM comprise nearly 35% of the bacterium's genome and are all repABC replicons. The repABC operons of the three largest plasmids of VF39SM were found to have strong incompatibility determinants in the non-protein coding regions. However, in all three repABC operons, the intergenic region between repB and repC was the strongest incompatibility factor; this intergenic region has been shown, for most repABC plasmids, to encode a counter-transcribed RNA (ctRNA) that regulates RepC abundance and therefore also rate of initiation of replication. To understand the way in which the ctRNA regulates replication and incompatibility, we carried out mutagenesis on this region from all three plasmids, using error-prone PCR. Mutants with altered incompatibility were detected by screening for their ability to co-exist in the same cell as the parent plasmid. Mutations that abolished the strong incompatibility phenotype were nearly all localized to the predicted ctRNA promoter regions. RT-PCR analysis confirmed that ctRNA was still produced in these promoter mutants, but transcriptional fusions of these mutated promoters to a gusA reporter gene showed a 10- to 50-fold decrease in activity when compared with the wild type promoter. For the repABC operons in this study, the intergenic region is critical in establishing incompatibility, and this appears to require a high level of transcription of the ctRNA.

An efficient plant regeneration and Agrobacterium-mediated genetic transformation of Tagetes erecta.

Tagetes erecta, L. an asteraceous plant of industrial and medicinal value, contains important compounds like pyrethrins, thiophenes and lutein, possessing immense potential for insecticidal, nematicidal and nutraceutical activities. Considering the importance and demand for these natural compounds, genetic manipulation of this crop for better productivity of secondary metabolites holds great significance. A rapid and reproducible direct regeneration and genetic transformation system is the prerequisite for genetic manipulation of any crop. This paper elucidates the establishment of an efficient direct regeneration and transformation protocol of T. erecta using Agrobacterium tumefaciens. Investigation of the effects of different types of explants (Hypocotyls, cotyledonary leaves, rachis and leaf sections) and different BAP and GA3 combinations on the regeneration frequency of T. erecta suggested that the best regeneration frequency (66%) with an average of 5.08 ± 0.09 shoot buds/explant was observed from hypocotyl explants cultured on media containing 1.5mg/l BAP and 5mg/l GA3. The transformation protocol was established using A. tumefaciens strain LBA4404, containing the binary vector pBI121, along with the gusA reporter gene with intron under the transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Various parameters like optimization of kanamycin concentration (200mg/l) for selection, standardization of cocultivation time (45min) and acetosyringone concentration (150μM) for obtaining higher transformation frequency were established using hypocotyl explants. The selected putative transgenic shoots were subsequently rooted on the Murashige and Skoog medium and transferred to the green house successfully. The plants were characterised by analysing the gus expression, amplification of 600bp npt II fragment and Southern blot hybridization using the PCR-amplified gusA fragment as probe. The standardised protocol established during the study will open new vistas for genetic manipulation and introduction of desired genes for genetic improvement of T. erecta.

Identification and molecular characterization of tissue-preferred rice genes and their upstream regularly sequences on a genome-wide level.

BackgroundGene upstream regularly sequences (URSs) can be used as one of the tools to annotate the biological functions of corresponding genes. In addition, tissue-preferred URSs are frequently used to drive the transgene expression exclusively in targeted tissues during plant transgenesis. Although many rice URSs have been molecularly characterized, it is still necessary and valuable to identify URSs that will benefit plant transformation and aid in analyzing gene function.ResultsIn this study, we identified and characterized root-, seed-, leaf-, and panicle-preferred genes on a genome-wide level in rice. Subsequently, their expression patterns were confirmed through quantitative real-time RT-PCR (qRT-PCR) by randomly selecting 9candidate tissue-preferred genes. In addition, 5 tissue-preferred URSs were characterized by investigating the URS::GUS transgenic plants. Of these URS::GUS analyses, the transgenic plants harboring LOC_Os03g11350 URS::GUS construct showed the GUS activity only in young pollen. In contrast, when LOC_Os10g22450 URS was used to drive the reporter GUS gene, the GUS activity was detected only in mature pollen. Interestingly, the LOC_Os10g34360 URS was found to be vascular bundle preferred and its activities were restricted only to vascular bundles of leaves, roots and florets. In addition, we have also identified two URSs from genes LOC_Os02G15090 and LOC_Os06g31070 expressed in a seed-preferred manner showing the highest expression levels of GUS activities in mature seeds.ConclusionBy genome-wide analysis, we have identified tissue-preferred URSs, five of which were further characterized using transgenic plants harboring URS::GUS constructs. These data might provide some evidence for possible functions of the genes and be a valuable resource for tissue-preferred candidate URSs for plant transgenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0331-2) contains supplementary material, which is available to authorized users.

Histone deacetylation modification participates in the repression of peanut (Arachis hypogaea L.) seed storage protein gene Ara h 2.02 during germination.

Genes encoding seed storage proteins (SSPs) are specifically and highly expressed during seed maturation. In Arabidopsis, chromatin-based mechanisms involved in the repression of SSPs during germination have been proposed. However, epigenetic regulation involved in repressing SSPs in vegetative tissues of peanut is not well understood. Histone deacetylase (HDAC) is a chromatin-remodelling factor that contributes to transcriptional repression in eukaryotes. To address whether histone deacetylation modification is involved in the repression of SSP genes during germination in peanut, we generated an Ara h 2.02pro : β-glucuronidase (GUS) construct by fusing the 1972bp Ara h 2.02 promoter of peanut (from -1972 to -1) to the GUS reporter gene and transformed it into wild-type Arabidopsis plants and HDAC mutants. GUS staining revealed that the mutation in HISTONE DEACETYLASE19 (HDA19) resulted in the ectopic expression of peanut SSP gene Ara h 2.02 in seedlings. In addition, Chromatin immunoprecipitation (ChIP) assays showed that the ectopic expression of Ara h 2.02 was accompanied by histone hyperacetylation during germination. These results suggest that histone deacetylation modification may play a vital role in repressing embryonic properties during the peanut vegetative growth.