Levels Of GUS Expression Research Articles

Isolation and Functional Characterization of a Constitutive Promoter in Upland Cotton (Gossypium hirsutum L.).

Multiple cis-acting elements are present in promoter sequences that play critical regulatory roles in gene transcription and expression. In this study, we isolated the cotton FDH (Fiddlehead) gene promoter (pGhFDH) using a real-time reverse transcription-PCR (qRT-PCR) expression analysis and performed a cis-acting elements prediction analysis. The plant expression vector pGhFDH::GUS was constructed using the Gateway approach and was used for the genetic transformation of Arabidopsis and upland cotton plants to obtain transgenic lines. Histochemical staining and a β-glucuronidase (GUS) activity assay showed that the GUS protein was detected in the roots, stems, leaves, inflorescences, and pods of transgenic Arabidopsis thaliana lines. Notably, high GUS activity was observed in different tissues. In the transgenic lines, high GUS activity was detected in different tissues such as leaves, stalks, buds, petals, androecium, endosperm, and fibers, where the pGhFDH-driven GUS expression levels were 3-10-fold higher compared to those under the CaMV 35S promoter at 10-30 days post-anthesis (DPA) during fiber development. The results indicate that pGhFDH can be used as an endogenous constitutive promoter to drive the expression of target genes in various cotton tissues to facilitate functional genomic studies and accelerate cotton molecular breeding.

Unveiling the defensive role of Snakin-3, a member of the subfamily III of Snakin/GASA peptides in potatoes.

The first in-depth characterization of a subfamily III Snakin/GASA member was performed providing experimental evidence on promoter activity and subcellular localization and unveiling a role of potato Snakin-3 in defense Snakin/GASA proteins share 12 cysteines in conserved positions in the C-terminal region. Most of them were involved in different aspects of plant growth and development, while a small number of these peptides were reported to have antimicrobial activity or participate in abiotic stress tolerance. In potato, 18 Snakin/GASA genes were identified and classified into three groups based on phylogenetic analysis. Snakin-1 and Snakin-2 are members of subfamilies I and II, respectively, and were reported to be implicated not only in defense against pathogens but also in plant development. In this work, we present the first in-depth characterization of Snakin-3, a member of the subfamily III within the Snakin/GASA gene family of potato. Transient co-expression of Snakin-3 fused to the green fluorescent protein and organelle markers revealed that it is located in the endoplasmic reticulum. Furthermore, expression analyses via pSnakin-3::GUS transgenic plants showed GUS staining mainly in roots and vascular tissues of the stem. Moreover, GUS expression levels were increased after inoculation with Pseudomonas syringae pv. tabaci or Pectobacterium carotovorum subsp. carotovorum and also after auxin treatment mainly in roots and stems. To gain further insights into the function of Snakin-3 in planta, potato overexpressing lines were challenged against P. carotovorum subsp. carotovorum showing enhanced tolerance to this bacterial pathogen. In sum, here we report the first functional characterization of a Snakin/GASA gene from subfamily III in Solanaceae. Our findings provide experimental evidence on promoter activity and subcellular localization and reveal a role of potato Snakin-3 in plant defense.

Identifying cis-Acting Elements Associated with the High Activity and Endosperm Specificity of the Promoters of Genes Encoding Low-Molecular-Weight Glutenin Subunits in Common Wheat (Triticum aestivum).

Low-molecular-weight glutenin subunits (LMW-GSs) associated with bread-baking quality and flour nutrient quality accumulate in endosperms of common wheat and related species. However, the mechanism underlying the expression regulation of genes encoding LMW-GSs has not been fully elucidated. In this study, we identified LMW-D2 and LMW-D7, which are highly and weakly expressed, respectively, via the analysis of RNA-sequencing data of Chinese Spring wheat and wheat transgenic lines transformed with 5' deletion promoter fragments and GUS fusion constructs. The 605-bp fragment upstream of the LMW-D2 start codon could drive high levels of GUS expression in the endosperm. The truncated endosperm box located at the -300 site resulted in the loss of LMW-D2 promoter activity, and a single-nucleotide polymorphism on the GCN4 motif was closely related to the expression of LMW-GSs. TCT and TGACG motifs, as well as the others located on the 5' distal end, might also be involved in the transcription regulation of LMW-GSs. In transgenic lines, fusion proteins of LMW-GS and GUS were deposited into protein bodies. Our findings provide new insights into the mechanism underlying the transcription regulation of LMW-GSs and will contribute to the development of wheat endosperm as a bioreactor for the production of nutraceuticals, antibodies, vaccines, and medicinal proteins.

An Agrobacterium-Mediated Transient Expression Method for Functional Assay of Genes Promoting Disease in Monocots.

Agrobacterium-mediated transient expression (AMTE) has been widely used for high-throughput assays of gene function in diverse plant species. However, its application in monocots is still limited due to low expression efficiency. Here, by using histochemical staining and a quantitative fluorescence assay of β-glucuronidase (GUS) gene expression, we investigated factors affecting the efficiency of AMTE on intact barley plants. We found prominent variation in GUS expression levels across diverse vectors commonly used for stable transformation and that the vector pCBEP produced the highest expression. Additionally, concurrent treatments of plants with one day of high humidity and two days of darkness following agro-infiltration also significantly increased GUS expression efficiency. We thus established an optimized method for efficient AMTE on barley and further demonstrated its efficiency on wheat and rice plants. We showed that this approach could produce enough proteins suitable for split-luciferase assays of protein-protein interactions on barley leaves. Moreover, we incorporated the AMTE protocol into the functional dissection of a complex biological process such as plant disease. Based on our previous research, we used the pCBEP vector to construct a full-length cDNA library of genes upregulated during the early stage of rice blast disease. A subsequent screen of the library by AMTE identified 15 candidate genes (out of ~2000 clones) promoting blast disease on barley plants. Four identified genes encode chloroplast-related proteins: OsNYC3, OsNUDX21, OsMRS2-9, and OsAk2. These genes were induced during rice blast disease; however, constitutive overexpression of these genes conferred enhanced disease susceptibility to Colletotrichum higginsianum in Arabidopsis. These observations highlight the power of the optimized AMTE approach on monocots as an effective tool for facilitating functional assays of genes mediating complex processes such as plant-microbe interactions.

Vascular dodecanoic acid of Arabidopsis mediates an insect resistance against Myzuspersicae.

The present study explores the compatible interaction between Arabidopsis thaliana and Myzus persicae to reduce host resistance from the previous aphid herbivore-mediated priming. The resumption of host resistance from the "reduced host resistance" was also recorded in due time when aphid herbivore was removed from leaf foliage. The vascular sap, isolated from the midpoint timing from the "reduced host resistance" to the "resumed host resistance" phase resolved in gas chromatography-mass spectrometry (GC-MS) analysis that identified an enrichment of dodecanoic acid (DA), an antibacterial metabolite and a saturated medium-chain fatty acid with a 12-carbon backbone. DA infiltration into leaf foliage revealed a significant reduction of aphid clonal proliferation on leaf foliage with concomitant reduction of the vascular microbiota titer as well as aphid body. The "resumed host resistance" from "reduced host resistance" also showed a comparable microbiota titer in comparison to control but the "reduced host resistance" evidenced a significant higher microbiota titer which was correlated with an enhanced aphid clonal proliferation on the leaf foliage. The DA infiltrated leaf foliage had no effect on total vascular sap ingestion by the aphid herbivore but induced RNA level of GUS expression under the control of promoter of pad-4, mpl-1, and sag-13. A similar pattern of gus expression was recorded from aphid herbivore. Thus, DA mediates aphid resistance towardaphid clonal proliferation in the host plant by manipulating vascular and aphid body microbiota titer.

Assessment of root-specific promoters in banana and tobacco and identification of a banana TIP2 promoter with strong root activity.

Genetic modification is one possible strategy to generate bananas (Musa spp.) with resistance to the soil-borne pathogen causing Fusarium wilt. The availability of banana root-specific promoters to target transgene expression to the sites of infection would be beneficial. We have assessed 18 promoter sequences derived from a range of plant species for their expression profiles in banana tissues to identify those with root-specific activity. Promoter sequences were isolated and fused to the β-glucuronidase (GUS) gene to assess their expression levels and tissue specificity in both banana and the model plant tobacco. Two heterologous promoters conferring high root expression levels in banana were identified, including a β‐glucosidase 1 (GLU1) promoter from maize and the RB7-type tonoplast intrinsic protein (TIP)-2 promoter from strawberry. Further, a novel Musa TIP2-2 promoter sequence was isolated and characterized which, when fused to the GUS gene, conferred very high GUS expression levels in banana roots. These promoters will expand the options for the control of gene expression in genetically modified bananas, providing a tool to develop plants with resistance not only to soil-borne diseases such as Fusarium wilt, but also for the improvement of other traits, such as nematode resistance, nutrition or abiotic stress resistance.

A Non-Canonical MITE in the WOX11 Promoter Is Associated with Robust Crown Root development in Rice.

The formation of tissues and organs in multicellular organisms is tightly controlled by transcriptional programs determined by temporal and spatial patterns of gene expression. As an important regulator of rice crown root development, WOX11 is essential for crown root formation and its transcript level is positively correlated with crown root biomass. However, how WOX11 is regulated during crown root primordium emergence and outgrowth still remains unknown. In this study, variations of the WOX11 genomic sequence were analyzed, and the highest genetic diversity was found within its promoter, which contained a non-canonical miniature inverted-repeat transposable element (ncMITE) sequence. Analysis of the WOX11 promoter-driven reporter gene GUS (β-glucuronidase) transgenic plants pWOX11(ncMITE+):GUS and pWOX11(ncMITE-):GUS uncovered higher GUS expression levels in crown roots of pWOX11(ncMITE+):GUS plants. Furthermore, pWOX11(ncMITE+):WOX11-FLAG in wox11 background could complement the crown root number and length compared to those of the wild type, while pWOX11(ncMITE-):WOX11-FLAG could not. These results suggested that the ncMITE was positively associated with WOX11 transcripts in rice crown roots. In addition, DNA methylation nearby the ncMITE region attenuated the activation effect of the ncMITE on WOX11 expression, which might also be the cause conferred to the root-specific expression of WOX11. This work provides novel insight into WOX11 expression regulation and reveals a promising target for genetic improvement of root architecture in rice.

SOCS-2 gene expression at diagnosis does not predict for outcome of chronic myeloid leukemia patients on imatinib treatment

SOCS-2 gene expression at diagnosis has been suggested as a predictor of clinical outcome in chronic myeloid leukemia (CML). In this study SOCS-2 and GUS expression levels were determined by real-time PCR in pretherapeutic samples at diagnosis. First, three patient groups were compared after assessment at 48 months: optimal molecular responders (n = 35), patients with resistance to imatinib (n = 28), and blast crisis patients (n = 27). A significant difference in SOCS-2 gene expression at diagnosis was observed comparing blast crisis vs. resistant patients (p = 0.042) and optimal responders (p = 0.010). Second, a validation sample of consecutively randomized patients (n = 123) was investigated. No discriminative SOCS-2 gene expression cutoff could be derived to predict molecular or cytogenetic response, progression-free or overall survival. Although SOCS-2 gene was differentially expressed at the time of diagnosis in blast crisis patients when compared to other groups, a prognostic impact in consecutively randomized patients was not observed.

WY195, a New Inducible Promoter From the Rubber Powdery Mildew Pathogen, Can Be Used as an Excellent Tool for Genetic Engineering.

Until now, there are few studies and reports on the use of endogenous promoters of obligate biotrophic fungi. The WY195 promoter in the genome of Oidium heveae, the rubber powdery mildew pathogen, was predicted using PromoterScan and its promoter function was verified by the transient expression of the β-glucuronidase (GUS) gene. WY195 drove high levels of GUS expression in dicotyledons and monocotyledons. qRT-PCR indicated that GUS expression regulated by the WY195 promoter was 17.54-fold greater than that obtained using the CaMV 35S promoter in dicotyledons (Nicotiana tabacum), and 5.09-fold greater than that obtained using the ACT1 promoter in monocotyledons (Oryza sativa). Furthermore, WY195-regulated GUS gene expression was induced under high-temperature and drought conditions. Soluble proteins extracted from WY195-hpaXm transgenic tobacco was bioactive. Defensive micro-HR induced by the transgene expression of hpaXm was observed on transgenic tobacco leaves. Disease resistance bioassays showed that WY195-hpaXm transgenic tobacco enhanced the resistance to tobacco mosaic virus (TMV). WY195 has great potential for development as a new tool for genetic engineering. Further in-depth studies will help to better understand the transcriptional regulation mechanisms and the pathogenic mechanisms of O. heveae.

Tissue-specific and stress-inducible promoters establish their suitability for containment of foreign gene(s) expression in transgenic potatoes.

The present study was conducted to determine efficiency of green tissue-specific (pRCA) and stress-inducible promoters (pRD29A) to express E. coli beta-glucuronidase (gusA) genein transgenic potatoes compared with constitutive promoter (35S CaMV). The promoter fragments were isolated from their original source and cloned upstream to gusA in pCAMBIA-1301 binary vector to develop plant expression constructs, i.e., pRCA-pCAMBIA and pRD29A-pCAMBIA. Agrobacterium strain GV2260 harboring recombinant plasmids were used to infect leaf discs and internodal explant of Lady Olympia cultivar. GUS histochemical analysis was performed at different stages to determine GUS activity in transgenic plants. To determine activity of stress-inducible promoter (pRD29A), transgenic plants were exposed to heat, drought and combination of both heat and drought stress. The real time (RT-qPCR) and GUS florimetric assays revealed that pRD29A promoter gets more activated under drought, heat and combination of both stresses. GUS expression levels were more than 10 folds high with pRD29A promoter compared to control. Likewise, the reduced transcripts levels of gusA gene under control of pRCA promoter were found in tuber/roots of transgenic plants compared to 35S promoter. GUS florimetric assays also showed decreased or no GUS expression in tubers. In conclusion, the results encourage the appropriate use of promoters to drive the expression of foreign gene(s) for the development of potato lines tolerant to biotic and abiotic stress while minimizing the risks of transgenic technology in potatoes.

An Efficient Agrobacterium-Mediated Genetic Transformation Using Embryonic Axis in Cotton (Gossypium hirsutum L.)

Agrobacterium-mediated genetic transformation approach allows for introducing novel genes in cotton (Gossypium hirsutum L.). Development of efficient regeneration and transformation protocol is very important for recalcitrant plants like cotton. In the present study, five-day-old germinated mature embryo parts especially embryonic axis, hypocotyl and plumule of cotton ‘Lashata’ were wounded and inoculated with A. tumefaciens strain GV2260 harbouring plasmid p35S-GUS-INT. The binary plasmid p35S-GUS-INT contains neomycin phosphotransferase II (NPTII) gene driven by nopaline synthase (NOS) promoter and β-glucuronidase (GUS) gene controlled by cauliflower mosaic virus (CaMV) 35S promoter. After inoculation, explants were co-cultivated in liquid and agar-solidified MS medium for 48 h in dark condition. Agar-solidified co-cultivation medium increased transformation frequency as compared to liquid medium. The putative primary transformants were confirmed with histochemical GUS assay, PCR and RT-PCR. However, comparing the three culture explant, the embryonic axis explants had significant difference with embryonic hypocotyl and plumule explants in terms of number and percentage (%) of GUS, PCR and RT-PCR positive plant. The total transformation efficiency was recorded as 3% with varying GUS expression levels.

Differential responses of root growth to nutrition with different ammonium/nitrate ratios involve auxin distribution in two tobacco cultivars

Nitrogen (N), the major forms of which are nitrate (NO3–) and ammonium (NH4+), plays an important role in plant growth and mediation of root development. However, the role of auxin in root growth in response to different NH4+/NO3– ratios remains unclear. Two tobacco cultivars (Nicotiana tabacum L.) were adopted in this study, which displayed variant growth features under the situations with sole NO3– nutrition ratio (NH4+/NO3– ratio: 0/100), low NO3– nutrition ratio (NH4+/NO3– ratio: 97/3), and optimal NH4+/NO3– ratio (50/50). We investigated the effects of the different NH4+/NO3– ratios on the formation and elongation of lateral roots (LRs), auxin concentration, DR5::GUS expression, 3H-labeled indole acetic acid ([3H]IAA) transport, and the expression of six PIN genes in tobacco roots. We also examined the effects of exogenous auxin and a transport inhibitor on LRs growth. The results are shown as follows, compared to optimal N nutrition conditions, the biomass and nitrogen (N) accumulation were largely reduced by sole and low NO3– nutrition treatment in NC89, but no difference was observed in Zhongyan 100. In most cases, sole and low NO3– nutrition impaired the elongation and formation of first-order lateral roots (1° LRs), only in NC89, thus reducing the root growth. IAA concentration and DR5::GUS expression levels decreased in roots when NC89 was subjected to sole and low NO3– nutrition media, suggesting that different NH4+/NO3– ratios affect the transport of auxin from leaves to roots. Results were similar following exogenous NAA application to low NO3– nutrition treated seedlings. Based on direct [3H]IAA transport measurement, the transport of polar auxin from shoots to roots decreased due to low NO3– nutrition. PIN4 expression levels were markedly decreased in roots of NC89 by sole and low NO3– nutrition, while they were unaffected in Zhongyan 100 roots. Overall, our findings suggest that LRs formation in tobacco seedlings is regulated by NH4+/NO3– ratios via modifying polar transport of auxin.

Development of Agrobacterium-mediated transient expression system in Caragana intermedia and characterization of CiDREB1C in stress response

BackgroundThe Agrobacterium-mediated transient transformation is a versatile and indispensable way of rapid analyzing gene function in plants. Despite this transient expression system has been successfully applied in a number of plant species, it is poorly developed in Caragana intermedia.ResultsIn this study, we established an Agrobacterium-mediated transient expression system in C. intermedia leaves and optimized the effect of different Agrobacterial strains, several surfactants and the concentration of Silwet L-77, which would affect transient expression efficiency. Among the 5 Agrobacterial strains examined, GV3101 produced the highest GUS expression level. Besides, higher level of transient expression was observed in plants infiltrated with Silwet L-77 than with Triton X-100 or Tween-20. Silwet L-77 at a concentration of 0.001% greatly improved the level of GUS transient expression. Real-time PCR showed that expression of CiDREB1C was highly up-regulated in transiently expressed plants and reached the highest level at the 2nd day after infiltration. Based on this optimized transient transformation method, we characterized CiDREB1C function in response to drought, salt and ABA treatment. The results showed that transiently expressed CiDREB1C in C. intermedia leaves could enhance the survival rate and chlorophyll content, and reduce the lodging rate compared with the control seedlings under drought, salt and ABA treatments. Furthermore, the rate of leaf shedding of CiDREB1C transient expression seedlings was lower than that of the control under ABA treatment.ConclusionsThe optimized transient expression condition in C. intermedia leaves were infiltrated with Agrobacterial strains GV3101 plus Silwet L-77 at a concentration of 0.001% added into the infiltration medium. Transiently expressed CiDREB1C enhanced drought, salt and ABA stress tolerance, indicated that it was a suitable and effective tool to determine gene function involved in abiotic stress response in C. intermedia.

Agroinfiltration is a key factor to improve the efficiency of apple and pear transformation

Genetic transformation of apple and pear is mainly performed via Agrobacterium tumefaciens. The average efficiency of transformation remains low in most laboratories and the success of the transformation experiments is very variable. Improving the efficiency and the reproducibility of apple and pear transformation is thus highly desirable. Adventitious regeneration ability of the explants is not a limiting factor for apple or pear transformation. The present study focuses on improving the frequency of stably transformed cells in the explants following A. tumefaciens inoculation. We report here the results of 36 independent transformation experiments on ‘Gala’ apple, performed with 10 different binary plasmids, comparing three methods of A. tumefaciens inoculation. Agroinfiltration (Agrobacterium-mediated vacuum infiltration) of the explants in a bacterial suspension containing a surfactant (Silwet L-77) at a low concentration (0.002% v/v) significantly increased the average transformation efficiency (mean rate of 5.8% and maximum rate of 30%) and reduced the number of failed experiments (16%) compared to the method of crushing the explants with non-traumatic forceps prior to immersion into the inoculum. The success of the agroinfiltration method was associated with a very high level of GUS expression one month after inoculation.In addition, agroinfiltration dramatically increased the transformation efficiency of pear, reaching rates of transformation between 50 and 80%, compared to inoculation with a scalpel dipped into the inoculum. Altogether, our results demonstrate that the production of large number of transgenic apple or pear lines in a short period of time is feasible using the agroinfiltration method.

Ethylene mediates dichromate-induced oxidative stress and regulation of the enzymatic antioxidant system-related transcriptome in Arabidopsis thaliana

Dichromate [Cr(VI)] induces phytotoxicity either directly by interfering in a plant’s physiological, biochemical, and/or molecular process or indirectly by stimulating reactive oxygen species accumulation (ROS). The underlying mechanism in Cr(VI)-induced ROS mediated phytotoxicity is elusive. In the current study, we investigate the possibility that Cr(VI)-induced alteration in the total biomass production, cell/tissue viability, and cell cycle-related transcriptome is mediated by the accumulation of high ROS level and that ethylene participates in this process. We showed that high Cr(VI) level reduces total biomass, chlorophyll contents, cell/tissue viability, and expression of cell cycle-related genes. Consistently, the oxidative damage and ROS accumulations were significantly increased in the seedlings exposed to higher Cr(VI) concentrations. Furthermore, Cr(VI)-induced EBS::GUS activity and expression level of ethylene signaling and biosynthesis related genes were also reported in the current study. Moreover, Cr(VI)-induced ROS accumulation by down-regulating enzymatic antioxidant system-related genes was slightly increased by the application of exogenous ethylene precursor (ACC), while significantly reduced by ethylene perception and biosynthesis antagonists silver (Ag) and cobalt (Co) respectively. Taken together, our results showed that ethylene mediates dichromate induced ROS accumulation by suppressing the expression level of enzymatic antioxidant enzymes-related genes.

Cloning and Functional Analysis of Phosphoethanolamine Methyltransferase Promoter from Maize (Zea mays L.).

Betaine, a non-toxic osmoprotectant, is believed to accumulate considerably in plants under stress conditions to maintain the osmotic pressure and promote a variety of processes involved in growth and development. Phosphoethanolamine N-methyltransferase (PEAMT), a key enzyme for betaine synthesis, is reported to be regulated by its upstream promoter. In the present investigation, by using the transgenic approach, a 1048 bp long promoter region of ZmPEAMT gene from Zea mays was cloned and functionally characterized in tobacco. Computational analysis affirmed the existence of abiotic stress responsive cis-elements like ABRE, MYC, HST, LST etc., as well as pathogen, wound and phytohormone responsive motifs. For transformation in tobacco, four 5′-deletion constructs of 826 bp (P2), 642 bp (P3), 428 bp (P4) and 245 bp (P5) were constructed from the 1048 bp (P1) promoter fragment. The transgenic plants generated through a single event exhibited a promising expression of GUS reporter protein in the leaf tissues of treated with salt, drought, oxidative and cold stress as well as control plants. The GUS expression level progressively reduced from P1 to P5 in the leaf tissues, whereas a maximal expression was observed with the P3 construct in the leaves of control plants. The expression of GUS was noted to be higher in the leaves of osmotically- or salt-treated transgenic plants than that in the untreated (control) plants. An effective expression of GUS in the transgenic plants manifests that this promoter can be employed for both stress-inducible and constitutive expression of gene(s). Due to this characteristic, this potential promoter can be effectively used for genetic engineering of several crops.

Auxin is Involved in Lateral Root Formation Induced by Drought Stress in Tobacco Seedlings

Drought stress is a common abiotic stress that affects plant root development. The goal of this study was to identify the role of auxin on aspects of root growth under water-deficient conditions in Nicotiana tabacum L. In this study, time-course development of lateral root (LR) growth in response to water deficiency (2.5% polyethylene glycol 6000, PEG) was analyzed in tobacco seedlings. LR formation in tobacco plants in response to drought stress depended on the experimental duration. First- and second-order LR formation was induced, respectively, by 43 and 28% on day 5 and by 57 and 40% on day 7 relative to control treatment. Thereafter, PEG-induced LR formation was not observed, and a decrease of 16% was recorded in first-order LR formation on day 11. A root pruning study suggested that PEG-induced LR formation plays a pivotal role in maintaining tobacco plant growth during drought stress. The change in the indole-3-acetic acid (IAA) concentration in leaves and roots was consistent with that of LR formation. This was corroborated by elevated DR5::GUS expression levels on day 5 and by decreased expression levels on day 9. Application of N-1-naphthylphthalamic acid (NPA) to PEG-treated seedlings reduced LR formation to levels similar to those of the control treatment; DR5::GUS expression was also similar between these treatments after NPA application. Results were similar following α-naphthylacetic acid application to control-treated seedlings. Measurement of [3H]IAA transport indicated that polar auxin transport from shoots to roots was increased by 43% under PEG application. Quantitative reverse transcription PCR revealed higher expression of YUC and PIN genes in PEG-treated plants, indicating that they play key roles in auxin synthesis and transport regulation in drought stress. Overall, these suggest that drought stress regulates LR formation in tobacco seedlings via modification of auxin synthesis and polar transport depending on the experimental stage.

Characterization of a strong green tissue-specific motif in rice photosystem I gene promoter Ppsak

Characterization of green tissue-specific promoters helps facilitate genetic improvement in crops. Here, we isolated a novel green tissue-specific expression gene Psak encoding photosystem I protein in rice through RT-PCR analysis. The 5′ flanking region from −635 to +60 (transcription start site of Psak as +1) covering parts of Psak gene and its promoter Ppsak was identified as be critical for green tissue-specific expression (including leaves and stems) in rice. Further promoter deletion analyses demonstrated that the promoter region from −559 to −232 was necessary and sufficient for green tissue-specific expression of Psak gene. Histochemical assays showed that the GUS expression of this 267-bp region was highly presented in leaves and stems, but reduced or absent in other tissues examined. The GUS expression level of this core promoter region could be 14.6- to 21.6-fold higher than that of the full-length region of Ppsak promoter in leaves and stems. These results combined with cis-acting elements prediction in Ppsak promoter suggested that the TCT motif located in this promoter played a key role in strong green tissue-specific expression.

Isolation and functional characterization of a novel stress inducible promoter from pigeonpea (Cajanus cajan L)

The present study deals with isolation and characterization of a novel hybrid-proline-rich protein gene (CcHyPRP) promoter from pigeonpea. Real time PCR analysis revealed that CcHyPRP expression was strongly induced by dehydration, salt, Abscisic acid (ABA) and Salicylic acid (SA) treatments. The CcHyPRP promoter, isolated by genome-walking method, contained 1112 bp and showed the presence of various cis-regulatory elements necessary for tissue specific expression and stress responsiveness. Different 5′ deletions of the promoter were generated and were used to drive the expression of β-glucuronidase reporter gene (gusA) in Arabidopsis thaliana. Histochemical and fluorometric assays confirmed that GUS expression driven by the full-length fragment (1112 bp) was higher when compared to different deletion fragments. Under normal conditions, GUS expression was predominantly detected in the roots and hypocotyls of transformants, while under mannitol, NaCl, ABA and SA treatment conditions higher GUS expression levels were observed in the roots and leaves. However, the GUS expression was mostly confined to the roots of transformants carrying 477 and 300 bp promoter regions. The results amply indicate that CcHyPRP promoter is regulated by different stress factors, and as such the promoter can be deployed in genetic engineering of crop plants for enhanced abiotic stress tolerance.

Optimization of Agrobacterium-mediated genetic transformation of shoot tip explants of green gram (Vigna radiata (L.) Wilczek)

Shoot tip explants prepared from seedlings of ML-267 genotype of green gram were inoculated on MSB5 medium supplemented with BAP (0–20 μM) individually or in combination with minimal concentration of auxins (NAA/IAA/IBA) for adventitious shoots formation. BAP alone without auxins was observed to be efficient in multiple shoot induction and optimum shoot proliferation was achieved on MSB5 medium containing 10 μM BAP with 100 % shoot induction frequency. 3-day-old explants gave best shoot multiplication response and the mean shoot number decreased significantly in 4-day and 5-day-old explants. The induced shoots rooted profusely on ½ MSB5 + 2.46 µM IBA and about 90 % of the plantlets survived after acclimatization and set seed normally. Shoot tip explants infected with A.tumefaciens (LBA4404) harboring pCAMBIA 2301 + AnnBj1 recombinant vector. Various factors which influence the competence of transformation were optimized based on the frequency of transient GUS expression in shoot tip explants. Optimum levels of transient GUS expression were recorded at pre-culture of explants for 2 days, infection for 10 min with Agro-culture of 0.8 OD and co-cultivation for 3 days on co-cultivation medium containing 100 µM acetosyringone in dark at 23 °C. Putative transformed shoots were produced on selection medium (shoot inductionmedium with100 mg/l kanamycin and 250 mg/l cefotaxim). PCR analysis confirmed the presence of AnnBj1, nptII, and uidA genes in T0 plants. Stable GUS activity was detected in flowers of T0 plants and leaves of T1 plants. PCR analysis of T1 progeny revealed AnnBj1 gene segregated following a Mendelian segregation pattern.