Expression Of GUS Gene Research Articles

Functional analysis of PagNAC045 transcription factor that improves salt and ABA tolerance in transgenic tobacco

BackgroundSalt stress causes inhibition of plant growth and development, and always leads to an increasing threat to plant agriculture. Transcription factors regulate the expression of various genes for stress response and adaptation. It’s crucial to reveal the regulatory mechanisms of transcription factors in the response to salt stress.ResultsA salt-inducible NAC transcription factor gene PagNAC045 was isolated from Populus alba×P. glandulosa. The PagNAC045 had a high sequence similarity with NAC045 (Potri.007G099400.1) in P. trichocarpa, and they both contained the same conserved motifs 1 and 2, which constitute the highly conserved NAM domain at the N-terminus. Protein-protein interaction (PPI) prediction showed that PagNAC045 potentially interacts with many proteins involved in plant hormone signaling, DNA-binding and transcriptional regulation. The results of subcellular localization and transient expression in tobacco leaves confirmed the nuclear localization of PagNAC045. Yeast two-hybrid revealed that PagNAC045 protein exhibits transcriptional activation property and the activation domain located in its C-terminus. In addition, the 1063 bp promoter of PagNAC045 was able to drive GUS gene expression in the leaves and roots. In poplar leaves and roots, PagNAC045 expression increased significantly by salt and ABA treatments. Tobacco seedlings overexpressing PagNAC045 exhibited enhanced tolerance to NaCl and ABA compared to the wild-type (WT). Yeast one-hybrid assay demonstrated that a bHLH104-like transcription factor can bind to the promoter sequence of PagNAC045.ConclusionThe PagNAC045 functions as positive regulator in plant responses to NaCl and ABA-mediated stresses.

Functional analysis of a viral promoter from a strawberry vein banding virus isolate from China

BackgroundPromoters are important factors affecting gene expression in cells. The driven activities of viral promoters were generally assessed to screen available promoters for transgenic and research and biotech industries. In this study, we cloned a full-length promoter from a Chinese isolate of strawberry vein banding virus (SVBV) and produced several deletion mutants for evaluation of applications in production of reporter proteins in stable transgenic plants.MethodsThe full-length promoter of SVBV (SP1) and its three deletion mutants (SP2, SP3, and SP4) were amplified using polymerase chain reaction. The effects of SVBV SP1, SP2, SP3, and SP4 on gene expression were evaluated using β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes.ResultsTransient expression assays showed that the SVBV SP1 promoter and its three deletion mutants all expressed the reporter genes, albeit at very different levels. Interestingly, transcriptional activity driven by the SP1 promoter was much higher than that of the cauliflower mosaic virus (CaMV) 35S promoter. After stable transformation of the GUS gene into Nicotiana tabacum plants, SVBV SP1-driven transgene expression was approximately 2.6-fold higher than CaMV 35S promoter-driven transgene expression. In addition, GUS gene expression levels were enhanced by co-inoculation of the plants with the SP1 promoter-driven vector carrying the GUS gene and the vector expressing SVBV open reading frame (ORF) V or ORF VI.ConclusionsThe SVBV SP1 promoter from the Chinese isolate evaluated in this study could successfully drive transient and stable expression in plants, it was a stronger promoter than the CaMV 35S and FLt-US promoters and may be more useful for the production of stable transgenic plants.

Expression of B. subtilis Phytase gene driven by fruit specific E8 promoter for enhanced minerals, metabolites and phytonutrient in cucumber fruit

The fruit nutrigenomics is an interesting and important research area towards nutrition enhancement. The phytic acid is one of the major antinutrient compound, present in seeded fruits and crops. It hinders the absorption of iron (Fe), zinc (Zn), magnesium (Mg), potassium (K) and calcium (Ca), causing mineral deficiencies. In the present study, the BsPhy gene was overexpressed in the cucumber fruits using the tomato fruit specific E8 and constitutive CaMV 35S promoter. The E8 promoter imparted heterologous expression of GUS gene in cucumber fruits, furthermore, the fruit specific expression of E8 promoter with BsPhy gene was confirmed in transgenics (E8::BsPhy) using anti rabbit-phytase antibody. The physio-biochemical analysis of transgenics revealed, maximum phytase activity in E8::BsPhy cucumber fruits at 10 days after anthesis (DAA) compared to 35S::BsPhy and wild-type (WT) fruits. Consequently, E8::BsPhy fruits also showed increased amount of inorganic phosphorus (Pi), total phosphorus (P), minerals (Zn, Fe, Mg, K, Ca), total carotenoid and other macronutrients at 10 DAA compared to 35S::BsPhy fruits. The metabolite profiling of fruits (10 DAA) showed increased sugars, amino acids, sugar acids and polyols, in both E8::BsPhy and 35S::BsPhy transgenics suggesting higher phytate metabolism, compared to WT fruits. Interestingly, both the transgenic fruits showed higher fruit biomass and yield along with improved nutritional quality, which can be attributed to increased P and Zn contents in transgenic fruits, compared to WT fruits. Our findings reveal that the BsPhy gene enhances minerals and macronutrients in transgenic cucumber fruits making it nutritious and healthy.

Isolation and functional validation of the VvZFP11 promoter associated with a signaling molecule and powdery mildew responses in grapevine

• Overexpression of VvZFP11 improves powdery mildew resistance in grapes. • The PME element of the VvZFP11 gene is essential for its function. • The VvZFP11 gene enables plants to acquire resistance by affecting the salicylic acid signaling pathway. VvZFP11 is up-regulated by powdery mildew (PM), methyl jasmonate (MeJA), and salicylic acid (SA) treatment, yet the underlying transcriptional regulatory mechanism remains unclear. Here, we demonstrate that a powdery mildew response element (PME) in the VvZFP11 promoter is important for countering the challenge to grapevine from PM, in which the defensive and stress response element (GTAAGGAAAC) is critical for specific responsiveness to SA, MeJA, and PM. Analysis of VvZFP11 promoter activity showed that VvZFP11 can activate GUS gene expression. SA induced VvZFP11 ’s expression, while VvZFP11 responded swiftly to PM. Analysis of cis- acting elements revealed that VvZFP11 contained diverse elements related to plant responses to biotic/abiotic stresses. Further experiments revealed PME in the VvZFP11 promoter was crucial for responding to PM. The PME and MV35S core promoters were fused and constructed into a single promoter, then inserted directionally into VvZFP11-GUS's upstream region to obtain a fusion construct. VvZFP11 transcript levels were significantly suppressed in transgenic plants with mutant PEM element after their inoculation with PM (based on Fig. 6). RT-qPCR results indicated the genes AtPR1, AtNPR1, AtPDF1.2 , and AtCOI , these related to SA and MeJA, were down-regulated in transgenic plants. The results also confirmed the defensive and stress response element in PME is a key component for the VvZFP11 promoter's function and that VvZFP11 enables plants to gain resistance to PM via the SA pathways.

Drought-Responsive NAC Transcription Factor RcNAC72 Is Recognized by RcABF4, Interacts with RcDREB2A to Enhance Drought Tolerance in Arabidopsis.

RcNAC72, a key transcription factor that may respond to drought stress in Rosa chinensis ‘Old Blush’, was selected in our previous study. In the present study, we found that RcNAC72 is localized in the nucleus and is a transcriptional activator. RcNAC72 expression could be significantly induced by drought, low temperature, salt as well as abscisic acid (ABA) treatment. Analysis of the promoter revealed that multiple abiotic stress and hormone response elements were located in the promoter region. The promoter could respond to drought, low temperature, salt and ABA treatments to activate GUS gene expression. Overexpressing RcNAC72 in Arabidopsis thaliana enhanced sensitivity to ABA and tolerance to drought stress. Silencing of RcNAC72 by virus-induced gene silencing (VIGS) in rose leaves significantly reduced leaf water loss tolerance and leaf extension capacity. Physical interaction of RcNAC72 with RcDREB2A was shown by means of the yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. RcABF4 was demonstrated to be able to bind to the promoter of RcNAC72 by means of the yeast one-hybrid (Y1H) assay. These results provide new insights into the regulatory network of RcNAC72 response to drought stress in roses.

Cloning and Functional Analysis of the Soybean GmRIQ2 Promoter

The RIQ gene can optimize the structure of light-harvesting complex II by regulating the degree of granum stacking, and ultimately participates in plant light stress responses. The GmRIQ2 gene contributes to plant photoprotection and may be involved in negative regulation of yield. To understand the regulatory mechanisms of GmRIQ2 expression, we isolated the GmRIQ2 promoter (PGmRIQ2) from the KenFeng 16 soybean genome. Sequence analysis showed that several cis-acting elements were involved in hormone responsiveness, seed-specific regulation, anaerobic induction and light responsiveness. PGmRIQ2 was expressed in soybean roots. GUS activity was detected in Arabidopsis thaliana rosette leaves, stems and roots, indicating that PGmRIQ2 tended to drive expression in rosette leaf vascular tissue and had spatiotemporal expression characteristics at the early growth stage. Three promoter deletion fragments of 450 bp, 284 bp and 194 bp were obtained by 5′ deletion. These three fragments all drove GUS gene expression in Arabidopsis thaliana rosette leaves, stems and roots. GUS gene expression decreased with deletion fragment truncation. In addition, the PGmRIQ2 auxin (IAA), abscisic acid (ABA) and methyl jasmonate (MeJA) response elements were located at base pairs −1661~−450, −450~−284 and −284~−194, respectively, and the promoter was induced by IAA, ABA, MeJA and light.

Overexpression of a Bcl-2-associated athanogene SlBAG9 negatively regulates high-temperature response in tomato

The Bcl-2-associated athanogene (BAG) gene is a multi-functional family of co-chaperones regulator, modulating plant stress response. Our previous study revealed that the SlBAG9 of tomato (Solanum lycopersicum) had the higher expression level induced by high-temperature (HT) at the transcriptional and protein levels, but its biological function was still unclear. Here, we conducted an in-depth analysis of SlBAG9. SlBAG9 protein was not located in the mitochondria but in the cytoplasm and nucleus. Many cis-acting elements involved in plant stress and hormone responses were located in the promoter regions of SlBAG9 including heat-shock element (HSE1). The β-glucuronidase (GUS) histochemical analysis showed that SlBAG9 promoter could drive GUS gene expression in transiently transformed Nicotiana tabacum leaves under non-inducing condition and HSE1 is critical for HT-induced GUS activity under HT. The transcription of SlBAG9 was expressed in different organs and was regulated by HT, cold, drought, and salt stresses as well as exogenous abscisic acid (ABA) and H2O2. To further elucidate SlBAG9 function in response to HT, the transgenic tomato plants overexpressing SlBAG9 were developed. Compared to the wild-type plants, SlBAG9-overexpressing plants exhibited more sensitivity to HT stress, reflected by the burning symptoms, the degradation of chlorophyll, and the reduction of photosynthetic rates. Additionally, SlBAG9-overexpressing lines showed higher accumulation of lipid peroxidation production (MDA) and H2O2, but lower activities of superoxide dismutase, catalase, and peroxidase. Therefore, it is speculated that SlBAG9 plays a negative role in thermotolerance probably by inhibition of antioxidant enzyme system leading to the oxidative damage, consequently aggravating the HT-caused injury phenotype.

Establishment of Agrobacterium tumefaciens – mediated genetic transformation of apple pathogen Marssonina coronaria using marker genes under the control of CaMV 35S promoter

Premature leaf fall of apple caused by Marssonina coronaria is economically very important apple disease and all the commercially available apple cultivars are susceptible to this disease. The non-availability of an efficient transformation system for this fungus hinders the functional genomics research. Herein, we report for the first time, the successful Agrobacterium-mediated transformation in apple leaf blotch fungus M. coronaria by transferring T-DNA harbouring the genes for hygromycin phosphotransferase (hpt), β-glucuronidase (uidA) and green fluorescent protein (gfp) under the control of CaMV 35S promoter. The key factors that affect the transformation efficiency including type of recipient fungal material, acetosyringone concentration, the conditions for co-cultivation, Agrobacterium concentration, Agrobacterium strains and membrane types as support were investigated. The present results have recommended that 250 μM concentration of acetosyringone, 24 °C temperature and 48 h time, 0.5 OD600 of A. tumefaciens, EHA105 Agrobacterium strain and Whatman filter paper were the optimal co-cultivation conditions for the transformation of M. coronaria by using fragmented mycelia suspension and mycelial plugs. We observed that conidia were tedious to transform as compared to the fragmented mycelia and mycelial plugs of this slow growing fungus. These optimized parameters yielded 54 and 70 average transformants per 60 mycelial plugs and 104 fragmented mycelia, respectively. Fungal transformants were analysed for T-DNA integration, gus gene expression and gfp gene expression. Strong gus histochemical staining and green fluorescence expression indicated that the CaMV 35S promoter can drive gene expression in M. croronaria. Some mutants showed difference in the morphology of the colony as compared to the wild type control. This report will be very useful to inspect molecular basis of apple-M. coronaria interactions by deciphering the functional roles of various genes in this pathogenic fungus.

Analysis of MeEf1A6 Gene Promoter Activity with In-vitro and In-vivo using Transient and Stable Expression Techniques in Tobacco Plant (Nicotiana tabacum)

The promoter is a part of the gene that functions in carrying out the gene expression, and its work activity becomes a matter of concern to ensure that expression works effectively. MeEF1A6 (Manihot esculenta Elongation Factor 1 Alfa - 6) is a promoter derived from cassava plants (Manihot esculenta). In previous studies, the MeEF1A6 promoter was successfully isolated, introduced, and characterized into the pBI121 plasmid, replacing the CaMV35S promoter. This study aims to analyze the activity of MeEF1A6 promoters in-vivo and in-vitro by using transient and transgenic techniques in tobacco plants. The pBI121 plasmid containing the MeEF1A6 promoter was introduced into Agrobacterium tumefaciens strain AGL1 and LBA4404. The promoter's work was then analyzed by the result of introducing it into the tobacco plant using the transient and stable transformation. The whole part of explants was used for transient study and tested in a minimum of two biological replicates. Sixty sheets of explant leaves that have been cut were used for stable transformation. The promoter work analysis was carried out with the GUS gene expression that integrated with the promoter with histochemical GUS assay. The transient produced a blue color in the roots, stems, and leaves on the whole repetition. The transverse incision in the stem shows the blue color on the epidermis and procambium tissue. Stable transformation using AGL1 as vector produced 43 shoots from 40 calli. A total of 43 shoots were selected with antibiotics and produced 27 plantlets that were successfully grown. Some plantlets are then reacted with x-gluc as histochemical GUS assay substrat and produced a blue color in the explants, indicating that the MeEF1A6 promoter has been successfully introduced. The results indicate that the MeEF1A6 promoter could work on plant tissue in roots, stems, leaves, and tissues that connect meristems such as procambium in tobacco plants. This reinforces the suspicion that the MeEF1A6 promoter performs work constitutionally as a constitutive promoter.

Overexpression Populusd-Type Cyclin Gene PsnCYCD1;1 Influences Cell Division and Produces Curved Leaf in Arabidopsis thaliana.

d-type cyclins (CYCDs) are a special class of cyclins and play extremely important roles in plant growth and development. In the plant kingdom, most of the existing studies on CYCDs have been done on herbaceous plants, with few on perennial woody plants. Here, we identified a Populus d-type cyclin gene, PsnCYCD1;1, which is mainly transcribed in leaf buds and stems. The promoter of PsnCYCD1;1 activated GUS gene expression and transgenic Arabidopsis lines were strongly GUS stained in whole seedlings and mature anthers. Moreover, subcellular localization analysis showed the fluorescence signal of PsnCYCD1;1-GFP fusion protein is present in the nucleus. Furthermore, overexpression of the PsnCYCD1;1 gene in Arabidopsis can promote cell division and lead to small cell generation and cytokinin response, resulting in curved leaves and twisted inflorescence stems. Moreover, the transcriptional levels of endogenous genes, such as ASs, KNATs, EXP10, and PHB, were upregulated by PsnCYCD1;1. Together, our results indicated that PsnCYCD1;1 participates in cell division by cytokinin response, providing new information on controlling plant architecture in woody plants.

Optimization of Agrobacterium Mediated Genetic Transformation in Paspalum scrobiculatum L. (Kodo Millet)

An efficient and reproducible protocol for Agrobacterium tumefaciens mediated genetic transformation was developed for kodo millet (Paspalum scrobiculatum L.) by optimizing various parameters. Agrobacterium strains EHA 105 and LBA 4404 harboring plasmids pCNL 56 and pCAMBIA 2300, respectively, provided the highest transformation efficiency. Addition of acetosyringone (AS) in infection medium (200 µM-EHA 105, 250 µM-LBA 4404) and co-cultivation medium (50 µM) increased the transformation efficiency. Transient and stable expression of gus gene was confirmed with histochemical assay of infected embryos and leaves of transformed plants, respectively. The best GUS response was obtained by pretreatment of callus with an antinecrotic mixture (10 mg/L Cys + 5 mg/L Ag + 2.5 mg/L As) at infection time of 20 min followed by co-cultivation for 3 days (EHA 105) and 5 days (LBA 4404) in dark. Regenerated transgenic plants were obtained after 8 to 10 weeks of selection on callus induction medium (NAA 0.5 mg/L, BAP 1 mg/L) containing 50 mg/L Kan + 250 mg/L Cef and were rooted for 2 weeks on MS medium containing PAA (1 mg/L) and phytagel. The plantlets established in greenhouse showed normal growth. Therefore, the protocol developed in the present study can be used for development of improved varieties of kodo millet.

Agrobacterium Mediated Transformation Optimizations for Sugarcane (Saccharum Officinarum L.) Cultivar SPF-234 with Direct Organogenesis

Sugarcane (Saccharum officinarum L.) is the most important food and energy crop worldwide. In the present study, an efficient Agrobacterium mediated transformation and regeneration system for sugarcane cultivar SPF-234 was established. Agrobacterium tumefaciens strains EHA101and LBA4404 using vector pIG121 Hm, having GUS, HPTII and NPTII genes were used. Polymerase chain reaction (PCR) and histochemical assays confirmed the GUS gene expression. A 620 bp fragment from GUS positive plants was amplified. The GUS expressing putative transformants were 35% of the total plants formed under 30 minute immersion time and 72 hr of incubation period. The co-cultivation media having 60 µM acetosyringone produced 66% GUS expressing plants for LBA4404 and 58% for EHA101. The maximum average number of directly produced shoot (59.5%) from leaf explant was in M6 media having 1.00 mg/l 6-Benzylaminopurine (BAP) and 2.5 mg/l Naphthaleneacetic acid (NAA). A significant decrease (17%) was observed when auxin (NAA) concentration was increased to 4.0 mg/l. The best response of shoot elongation was observed in SE4 media having equal concentration (2.00 mg/l) of both kinetin and BAP. Increased concentrations of kinetin significantly decreased shoot elongation of the subject cultivar. Agrobacterium strain LBA4404 performed better for genetic transformation of the said sugarcane cultivar.This quick and less expensive transformation and direct regeneration system could be exploited for sugarcane on commercial scale in general, and for this elite cultivar in particular.

Efficient Agrobacterium tumefaciens-mediated stable genetic transformation of green microalgae, Chlorella sorokiniana.

The online version contains supplementary material available at 10.1007/s13205-021-02750-7.

The ethylene-responsive transcription factor of durum wheat, TdSHN1, confers cadmium, copper, and zinc tolerance to yeast and transgenic tobacco plants.

Cadmium (Cd), copper (Cu), and zinc (Zn) are among the most common heavy metals (HMs) present in polluted soils. While some HMs are required for key biological processes, they are toxic when present in excess. This toxicity damages plant health, decreases crop yields, and can impact human health via the food chain. For example, durum wheat is a staple food that is known to accumulate Cd when grown on polluted soils. Plant response to HM stress is complex and involves several transcription factors (TFs) among which members of the ERF family. Although roles of SHINE-type ERF transcription factors in abiotic stress tolerance have been thoroughly investigated, there is little information concerning their role in HM stress tolerance. In the present study, we investigated the role of durum wheat TdSHN1 TF in HM response and tolerance. Results showed that TdSHN1 expression was strongly induced by Cd, Cu, and Zn in durum wheat seedlings. In addition, TdSHN1 gene promoter directed HM-inducible GUS gene expression in transgenic tobacco. Overexpression of TdSHN1 encoding cDNA in transgenic yeast and tobacco conferred Cd, Cu, and Zn tolerances. Interestingly, transgenic tobacco lines exhibited longer roots and greater biomass accumulation, retained more chlorophyll, and produced less ROS than WT plants, when subjected to excess HMs. In addition, transgenic tobacco lines had higher activities of ROS-scavenging enzymes (SOD and CAT) which might have contributed to their HM tolerance. This study suggested that TdSHN1 is a potential candidate for improving HM tolerance in plants and phytoremediation of HM-contaminated soils.

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.

Establishment of the 'imbibed seed piercing' method for Agrobacterium-mediated transformation of jute and flax bast fibre crops via phloem-specific expression of the β-glucuronidase Gene

Jute (Corchorus capsularis) and flax (Linum usitatissimum) have been established as important sources of bast fibres (phloem fibres) worldwide. These long and lustrous natural fibres are used for various commercial and industrial purposes and hold immense future prospects. To meet the worldwide demand for bast fibres, improvement in the production and quality of jute and flax is essential. Molecular breeding is an expeditious approach for varietal improvement and requires an efficient gene transformation system. The available methods for jute and flax transformation are highly specific to species and variety, requiring a long time and rigorous preparation of media, explants, and screening procedures. In this study, we used a fast and stable method for Agrobacterium tumefaciens-mediated transformation of jute (variety JRC321) and flax (variety FT-897), known as the ‘imbibed seed piercing’ method (ISPM), where seeds were used as explants. A phloem-specific Arabidopsis sucrose-proton H+ symporter 2 (AtSUC2) promoter-driven gus gene (β-glucuronidase) was used for the assessment of genetic transformation of jute and flax plants. Transformed seedlings were selected following three consecutive selection pressure tests conducted at two-week intervals using kanamycin-supplemented culture media. Transgene integration was verified using Southern blotting and PCR-based molecular characterization of jute and flax plants. The gus gene expression levels in the transgenic jute and flax tissue samples were analysed by semi-quantitative and quantitative RT-PCR. The GUS protein expression in transgenic plant tissues was confirmed by the development of blue colour in the histochemical GUS assay. Blue colouration was observed only in phloem cells of the tested plant tissues. Transgenic progeny plants of subsequent generations followed the 3:1 transgene segregation pattern and exhibited phloem-specific GUS expression. This fast, easy, and stable ISPM could also be applied to other fibre producing industrial crops and could form a part of a modern CRISPR/Cas9-based genome editing tool by efficiently delivering CRISPR vectors in jute and flax plants.

The cold response regulator CBF1 promotes Arabidopsis hypocotyl growth at ambient temperatures.

Light and temperature are two core environmental factors that coordinately regulate plant growth and survival throughout their entire life cycle. However, the mechanisms integrating light and temperature signaling pathways in plants remain poorly understood. Here, we report that CBF1, an AP2/ERF-family transcription factor essential for plant cold acclimation, promotes hypocotyl growth under ambient temperatures in Arabidopsis. We show that CBF1 increases the protein abundance of PIF4 and PIF5, two phytochrome-interacting bHLH-family transcription factors that play pivotal roles in modulating plant growth and development, by directly binding to their promoters to induce their gene expression, and by inhibiting their interaction with phyB in the light. Moreover, our data demonstrate that CBF1 promotes PIF4/PIF5 protein accumulation and hypocotyl growth at both 22°C and 17°C, but not at 4°C, with a more prominent role at 17°C than at 22°C. Together, our study reveals that CBF1 integrates light and temperature control of hypocotyl growth by promoting PIF4 and PIF5 protein abundance in the light, thus providing insights into the integration mechanisms of light and temperature signaling pathways in plants.

Expression of gus and gfp genes in amphidiploid spelt wheat (Triticum spelta L.) after Agrobacterium-mediated transformation

Мета. Дослідити експресію генів gus та gfp в калюсних експлантах амфідиплоїдної пшениці спельти ( Triticum spelta L.) після Agrobacterium -опосередкованої генетичної трансформації. Методи. Для трансформації було обрано сорт пшениці спельти озимої ‘Європа’. Як експланти використовували калюси, отримані зі зрілих зародків. Прекультивацію калюсів здійснювали на живильному середовищі МС (Мурасіге–Скуга), доповненому 2 мг/л 2,4-Д (2,4-дихлорфеноксиоцтова кислота) та 10 мг/л нітратом срібла. Для генетичної трансформації використовували Agrobacterium tumefaciens Conn., штам GV3101, та генетичну конструкцію з репортерними генами gus (ген бета-глюкуронідази (-glucuronidase)) та gfp (ген зеленого флюоресцентного білка (Green Fluorescent Protein, GFP)). Калюси трансформували шляхом інокуляції з агробактеріями та вакуумною інфільтрацією. Далі їх ко-культивували на середовищі МС із 2 мг/л 2,4-Д та 10 мг/л AgNO 3 , але без антибіотиків. Експресію гена gus перевіряли за допомогою гістохімічного, а гена gfp – візуального аналізу (флуоресценція білка GFP в УФ світлі). Рівні експресії генів gfp та gus оцінювали за допомогою програмного забезпечення ImajeJ. Інтеграцію генів gfp та gus в геном спельти перевіряли методом ПЛР . Результати. Генетична трансформація калюсних експлан­тів спельти шляхом їхньої інокуляції в живильному середовищі з агробактеріями та вакуумною інфільтрацією відбувалась з різною частотою. Рівень експресії гена gus за вакуумної інфільтрації становив 4,66 ± 0,74%, за інокуляції – 4,00 ± 0,91%; а гена gfp за вакуумної інфільтрації – 3,66 ± 0,74%, за інокуляції – 4,66 ± 1,39%. Рівень експресії гена gfp був вищим у разі використання інокуляції з агробактеріями, а гена gus – при ва­ку­умній інфільтрації. За допомогою ПЛР-аналізу було під­тверджено інтеграцію генів gfp та gus в геном калюсів спельти. Довжина ПЛР продукту із праймерами до гена gus становила 240 п. н., а до гена gfp – 717 п. н. Висновки. Використання методів вакуумної інфільтрації та інокуляції для генетичної трансформації спельти дали різні результати. Частота генетичної трансформації коливалась від 3,66 до 4,66%. Agrobacterium -опосередкована генетична трансформація амфідиплоїдної пшениці спельти дозволяє дослідити експресію репортерних генів gus та gfp за використання калюсних експлантів, отриманих із зрілих зародків.

Reliable and Efficient Agrobacterium tumefaciens-Mediated Genetic Transformation of Dianthus spiculifolius

Dianthus spiculifolius is a perennial herbaceous flower with strong environmental adaptability and is an important ornamental ground cover plant. In this study, seeds of D. spiculifolius were used as explants for callus induction, adventitious bud differentiation, and rooting by adding different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyl aminopurine (6-BA), and naphthaleneacetic acid (NAA) to Murashige and Skoog medium. The calli generated were co-cultured with Agrobacterium tumefaciens EHA105 containing pBI121-GUS or pBI121-GFP plasmids for 30 min, and transgenic regenerated plants were obtained by kanamycin (30 mg · L−1) screening. RT-PCR confirmed the stable expression of the exogenous GUS and GFP genes in the D. spiculifolius. The β-glucuronidase (GUS) histochemical staining confirmed GUS gene expression in transgenic calli, adventitious buds, and regenerated plants of D. spiculifolius. The green fluorescent protein (GFP) visual analysis showed GFP gene expression in transgenic calli. Furthermore, subcellular localization analysis showed that the three organelle marker proteins were not only successfully expressed but also accurately localized to their corresponding organelles in D. spiculifolius callus cells. These results indicated a successful establishment of a reliable and efficient A. tumefaciens-mediated genetic transformation system, which will contribute to functional gene research and genetic improvement of D. spiculifolius.

Two promoter regions confer heat-induced activation of SlDREBA4 in Solanum lycopersicum

Dehydration-responsive element binding (DREB) transcription factors activate the expression of downstream functional genes in combination with a dehydration-responsive element (DRE), and thereby improve the resistance of plants to abiotic stresses such as heat. However, the upstream regulatory mechanism of DREB genes under heat is unclear. A DREBA4 subfamily transcription factor (SlDREBA4), which is heat-responsive and improves heat resistance, was isolated from Solanum lycopersicum ‘Microtom’. In this study, promoter truncation experiments were performed to verify changes in β-glucuronidase (GUS) enzyme activity and GUS gene expression levels in transgenic plants with different lengths of promoter fragments under heat and to identify specific regions in the promoter that respond to heat. Our results showed that the GUS reporter gene was constitutively expressed in tissues of the full-length promoter transgenic ‘Microtom’ plants, with higher expression in conducting tissues of root, stem, and leaf, as well as sepals of flowers and fruits. Under heat treatment, GUS enzyme activity and GUS gene expression levels in tissues of the full-length promoter transgenic plants increased. Promoter deletion analysis identified two positive regulatory regions (−1095 to −730 bp and −162 to −38 bp) responsible for the promoter’s response to heat. These results indicated that the heat shock element (HSE) and MYC recognition sequences may cooperate in heat-induced activation of SlDREBA4 promoter.