Promoter Elements Research Articles

Engineering a carbon source-responsive promoter for improved biosynthesis in the non-conventional yeast Kluyveromyces marxianus

Many desired biobased chemicals exhibit a range of toxicity to microbial cell factories, making industry-level biomanufacturing more challenging. Separating microbial growth and production phases is known to be beneficial for improving production of toxic products. Here, we developed a novel synthetic carbon-responsive promoter for use in the rapidly growing, stress-tolerant yeast Kluyveromyces marxianus, by fusing carbon-source responsive elements of the native ICL1 promoter to the strong S. cerevisiae TDH3 or native NC1 promoter cores. Two hybrids, PIT350 and PIN450, were validated via EGFP fluorescence and demonstrated exceptional strength, partial repression during growth, and late phase activation in glucose- and lactose-based medium, respectively. Expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for synthesis of the polyketide triacetic acid lactone (TAL) under the control of PIN450 increased TAL more than 50% relative to the native NC1 promoter, and additional promoter engineering further increased TAL titer to 1.39 g/L in tube culture. Expression of the Penicillium griseofulvum 6-methylsalicylic acid synthase (6-MSAS) under the control of PIN450 resulted in a 6.6-fold increase in 6-MSA titer to 1.09 g/L and a simultaneous 1.5-fold increase in cell growth. Finally, we used PIN450 to express the Pseudomonas savastanoi IaaM and IaaH proteins and the Salvia pomifera sabinene synthase protein to improve production of the auxin hormone indole-3-acetic acid and the monoterpene sabinene, respectively, both extremely toxic to yeast. The development of carbon-responsive promoters adds to the synthetic biology toolbox and available metabolic engineering strategies for K. marxianus, allowing greater control over heterologous protein expression and improved production of toxic metabolites.

The Public Health Approach to Oral Health: A Literature Review

Background: Oral health (OH) has evolved beyond dental concerns to encompass psychosocial dimensions and overall well-being. This study reviews OH strategies within a public health framework to identify key elements for effective OH promotion. Methods: A literature review following PRISMA guidelines identified 42 relevant articles from 62 screened. Five themes emerged: group-level, individual-level, policy-level (emphasizing devising OH policies), healthcare delivery, and communication. Common components included OH education, behaviour change, access to OH services, and policy integration. Results: Thematic analysis identified five overarching themes in oral health (OH) promotion strategies, with a focus on tailored approaches for specific populations and components such as education, access to services, interventions, and policy, emphasizing the multifaceted nature of OH promotion. Conclusion: Effective OH promotion requires a multifaceted approach and tailored strategies with interprofessional collaboration. Future research should focus on cost-effectiveness and user-friendly resources for OH professionals and policymakers.

Comprehensive analysis of the bZIP gene family in ‘Duli’ pear (Pyrus betulifolia Bunge): Insights for research on abiotic stress, especially drought, in pear

Plants are unavoidably subjected to various abiotic stresses, such as drought, salt, and cold, during their growth and development. Drought, accompanied by water shortages in North China, has become an environmental factor seriously affecting agriculture production. Basic leucine zipper (bZIP) is one of the most diverse transcription factor families in plants. It participates in the regulation of many physiological processes and plays an important role in plants’ response to abiotic stress. In this study, for the first time, 88 bZIP family members (PbbZIPs) in ‘Duli’ pear (Pyrus betulifolia Bunge) were identified using both hidden Markov model (HMM) and basic local alignment search tool (BLAST) methods. A phylogenetic tree of all the PbbZIPs members was constructed. The physicochemical properties, subcellular localisation, chromosomal location, gene structure, conserved motifs and domains, secondary and tertiary structures, and cis-acting promoter elements of the 88 family members were comprehensively analysed. The results indicated that PbbZIPs may play relevant roles in stress response in ‘Duli’. Therefore, we determined the expressions of all PbbZIPs in ‘Duli’ seedlings under drought, salt, and cold conditions, and in seedlings treated with stress-related hormones such as abscisic acid (ABA), salicylic acid (SA), methyl jasmonate (MeJA), and melatonin (MT). PbbZIPs responsive to various abiotic stresses and hormones were screened. In addition, we determined the expression patterns of 12 PbbZIP genes that strongly responded to both drought and ABA in ‘Duli’ tissue culture plants treated with different concentrations of PEG4000 at different time points. Finally, we selected three key genes (PbbZIP65, PbbZIP77 and PbbZIP81) that may play a major role in drought resistance in ‘Duli’, and preliminarily identified their function through the VIGS method, finding that PbbZIP77 could positively regulate the drought resistance of ‘Duli’ seedlings, while PbbZIP65/81 could negatively regulate the drought resistance of ‘Duli’ seedlings. This study not only broadens our knowledge about the bZIP gene family in ‘Duli’ pear, but also provides notable guidance for further functional research on PbbZIPs in abiotic stress, especially drought resistance, in the pear plant.

RsRbohD1 Plays a Significant Role in ROS Production during Radish Pithiness Development.

Pithiness is one of the physiological diseases of radishes, which is accompanied by the accumulation of reactive oxygen species (ROS) during the sponging of parenchyma tissue in the fleshy roots. A respiratory burst oxidase homolog (Rboh, also known as NADPH oxidase) is a key enzyme that catalyzes the production of ROS in plants. To understand the role of Rboh genes in radish pithiness, herein, 10 RsRboh gene families were identified in the genome of Raphanus sativus using Blastp and Hmmer searching methods and were subjected to basic functional analyses such as phylogenetic tree construction, chromosomal localization, conserved structural domain analysis, and promoter element prediction. The expression profiles of RsRbohs in five stages (Pithiness grade = 0, 1, 2, 3, 4, respectively) of radish pithiness were analyzed. The results showed that 10 RsRbohs expressed different levels during the development of radish pithiness. Except for RsRbohB and RsRbohE, the expression of other members increased and reached the peak at the P2 (Pithiness grade = 2) stage, among which RsRbohD1 showed the highest transcripts. Then, the expression of 40 genes related to RsRbohD1 and pithiness were analyzed. These results can provide a theoretical basis for improving pithiness tolerance in radishes.

Genome-wide identification and expression analysis of Ubiquitin-specific protease gene family in maize (Zea mays L.)

BackgroundUbiquitin-specific proteases (UBPs) are a large family of deubiquitinating enzymes (DUBs). They are widespread in plants and are critical for plant growth, development, and response to external stresses. However, there are few studies on the functional characteristics of the UBP gene family in the important staple crop, maize (Zea mays L.).ResultsIn this study, we performed a bioinformatic analysis of the entire maize genome and identified 45 UBP genes. Phylogenetic analysis indicated that 45 ZmUBP genes can be divided into 15 subfamilies. Analysis of evolutionary patterns and divergence levels indicated that ZmUBP genes were present before the isolation of dicotyledons, were highly conserved and subjected to purifying selection during evolution. Most ZmUBP genes exhibited different expression levels in different tissues and developmental stages. Based on transcriptome data and promoter element analysis, we selected eight ZmUBP genes whose promoters contained a large number of plant hormones and stress response elements and were up-regulated under different abiotic stresses for RT-qPCR analysis, results showed that these genes responded to abiotic stresses and phytohormones to varying degrees, indicating that they play important roles in plant growth and stress response.ConclusionsIn this study, the structure, location and evolutionary relationship of maize UBP gene family members were analyzed for the first time, and the ZmUBP genes that may be involved in stress response and plant growth were identified by combining promoter element analysis, transcriptome data and RT-qPCR analysis. This study informs research on the involvement of maize deubiquitination in stress response.

Auxin efflux carrier PsPIN4 identified through genome-wide analysis as vital factor of petal abscission.

PIN-FORMED (PIN) proteins, which function as efflux transporters, play many crucial roles in the polar transportation of auxin within plants. In this study, the exogenous applications of auxin IAA and TIBA were found to significantly prolong and shorten the florescence of tree peony (Paeonia suffruticosa Andr.) flowers. This finding suggests that auxin has some regulatory influence in petal senescence and abscission. Further analysis revealed a total of 8 PsPINs distributed across three chromosomes, which could be categorized into two classes based on phylogenetic and structural analysis. PsPIN1, PsPIN2a-b, and PsPIN4 were separated into the "long" PIN category, while PsPIN5, PsPIN6a-b, and PsPIN8 belonged to the "short" one. Additionally, the cis-regulatory elements of PsPIN promoters were associated with plant development, phytohormones, and environmental stress. These genes displayed tissue-specific expression, and phosphorylation sites were abundant throughout the protein family. Notably, PsPIN4 displayed distinct and elevated expression levels in roots, leaves, and flower organs. Expression patterns among the abscission zone (AZ) and adjacent areas during various flowering stages and IAA treatment indicate that PsPIN4 likely influences the initiation of peony petal abscission. The PsPIN4 protein was observed to be co-localized on both the plasma membrane and the cell nucleus. The ectopic expression of PsPIN4 reversed the premature flower organs abscission in the Atpin4 and significantly protracted florescence when introduced to Col Arabidopsis. Our findings established a strong basis for further investigation of PIN gene biological functions, particularly concerning intrinsic relationship between PIN-mediated auxin polar.

Instagram Ads Usage to Increase Interest of Generation Z House Buying of PT Wengga Citra Jaya in East Kotawaringin

Social media has become a digital platform for carrying out marketing activities. One of the social media used to carry out marketing activities is Instagram through the Instagram Ads feature. With a wise advertising strategy and utilizing existing features, Instagram Ads can be a very effective tool in influencing consumer buying interest and driving business growth. This research aims to analyze the use of Instagram Ads in an effort to increase interest in buying a house from PT Wengga Citra Jaya in East Kotawaringin Regency among generation Z. Generation Z's interest in buying a house is still relatively low due to several reasons and considerations. Generation Z are potential buyers in the future, so the low buying interest in generation Z must be overcome by carrying out marketing communication activities. This research uses qualitative method by collecting data through interviews with sources who are responsible for PT Wengga Citra Jaya's marketing activities and interviews with experts. The results of this research show that the efforts to use Instagram Ads carried out by PT Wengga Citra Jaya have not been optimal because there are elements that have not been used optimally. For maximum use, Instagram Ads must have information, images, audio, price and promotional elements. This research can be a reference for business actors who want to take advantage from Instagram Ads. To support maximum use of Instagram Ads, the supporting elements of the Instagram Ads should be maximally utilized to get more optimal results.

Analisis Strategi Pemasaran yang Digunakan oleh Usaha Mikro Mogobo Snack Tuban untuk Meningkatkan Volume Penjualan

This research is intended to evaluate the marketing strategy implemented by the Mogobo Snack Tuban Micro Enterprise to increase their sales. Mogobo Snack is a micro business that produces fried meatball chips with a spicy taste as its characteristic. This research adopts qualitative methods using observation, interviews and documentation instruments. The findings from the research show that the Mogobo Snack Tuban Micro Enterprise implements a marketing strategy in the form of a marketing mix consisting of product, price, promotion and distribution elements. The strategy includes production of high-quality chips, pricing based on production costs, online and offline marketing, as well as promotion via social media and a door-to-door approach. The SWOT analysis shows several alternative strategies that Mogobo Snack can implement, such as increasing digital marketing, developing new products, collaborating with culinary influencers, and expanding into traditional markets. By implementing these strategies, Mogobo Snack can overcome the challenges of competition and changing consumer trends, thereby increasing sales volume and increasing its market share and profitability.

EPAS1, a hypoxia- and ferroptosis-related gene, promotes malignant behaviour of cervical cancer by ceRNA and super-enhancer.

Hypoxia and Ferroptosis are associated with the malignant behaviour of cervical cancer. Endothelial PAS domain-containing protein 1 (EPAS1) contributes to the progression of cervical cancer. EPAS1 plays important roles in hypoxia and ferroptosis. Using the GEO dataset, machine-learning algorithms were used to screen for hypoxia- and ferroptosis-related genes (HFRGs) in cervical cancer. EPAS1 was identified as the hub gene. qPCR and WB were used to investigate the expression of EPAS1 in normal and cervical cancer tissues. The proliferation, invasion and migration of EPAS1 cells in HeLa and SiHa cell lines were detected using CCK8, transwell and wound healing assays, respectively. Apoptosis was detected by flow cytometry. A dual-luciferase assay was used to analyse the MALAT1-miR-182-5P-EPAS1 mRNA axis and core promoter elements of the super-enhancer. EPAS1 was significantly overexpressed in cervical cancer tissues. EPAS1 could increase the proliferation, invasion, migration of HeLa and SiHa cells and reduce the apoptosis of HeLa and SiHa cell. According to the double-luciferase assay, EPAS1 expression was regulated by the MALAT1-Mir-182-5p-EPAS1 mRNA axis. EPAS1 is associated with super-enhancers. Double-luciferase assay showed that the core elements of the super-enhancer were E1 and E3. EPAS1, an HFRG, is significantly overexpressed in cervical cancer. EPAS1 promotes malignant behaviour of cervical cancer cells. EPAS1 expression is regulated by super-enhancers and the MALAT1-miR-182-5P- EPAS1 mRNA axis. EPAS1 may be a target for the diagnosis and treatment of cervical cancer.

Genome-Wide Identification of GAST Family Members and Their Potential Roles in Epicotyl Dormancy in Chinese Cork Oak (Quercus variabilis).

Chinese cork oak (Quercus variabilis Blume) is a widespread tree species with high economic and ecological values. Chinese cork oak exhibits epicotyl dormancy, causing emergence heterogeneity and affecting the quality of seedling cultivation. Gibberellic acid-stimulated transcript (GAST) is a plant-specific protein family that plays a crucial regulatory role in plant growth, development, and seed germination. However, their evolution in Chinese cork oak and roles in epicotyl dormancy are still unclear. Here, a genome-wide identification of the GAST gene family was conducted in Chinese cork oak. Ten QvGAST genes were identified, and nine of them were expressed in seed. The physicochemical properties and promoter cis-acting elements of the selected Chinese cork oak GAST family genes indicated that the cis-acting elements in the GAST promoter are involved in plant development, hormone response, and stress response. Germinated seeds were subjected to gibberellins (GAs), abscisic acid (ABA), and fluridone treatments to show their response during epicotyl dormancy release. Significant changes in the expression of certain QvGAST genes were observed under different hormone treatments. QvGAST1, QvGAST2, QvGAST3, and QvGAST6 exhibited upregulation in response to gibberellin. QvGAST2 was markedly upregulated during the release of epicotyl dormancy in response to GA. These findings suggested that QvGAST2 might play an important role in epicotyl dormancy release. This study provides a basis for further analysis of the mechanisms underlying the alleviation of epicotyl dormancy in Chinese cork oak by QvGASTs genes.

Product Branding and Customer Patronage of Champion Breweries Plc’s Products, Uyo, Akwa Ibom State, Nigeria

The study examined the effect of product branding on customers’ patronage of Champion Breweries products in Uyo, Akwa Ibom State. The research design adopted was a survey research in which 400 questionnaires were administered to the respondents, out of which 361 were duly filled and returned. Two hypotheses guided the study while regression analysis was used to analyse the data collected. Findings revealed, amongst others, that brand awareness (R2 = 0.885, P<0.05) and brand image (R2= 0.958, P=0.000) have a positive and significant effect on customers’ patronage of Champion Breweries’ products. Based on the findings, it was concluded amongst others, that branding significantly affects customers’ patronage of Champion Breweries products in Uyo. From the findings and conclusion, the study recommended that the Management of Champion Breweries, Uyo should endeavour to expose their brands to their target market using appropriate promotional mix elements to enhance brand awareness and brand image as the patronage of Champion Breweries Products is determined by them.

A bZIP type transcription factor from Picrorhiza kurrooa modulates the expression of terpenoid biosynthesis genes in Nicotiana benthamiana

bZIP family of transcription factors are DNA-binding proteins which bind ACGT motifs in the upstream promoter region of genes. bZIP proteins play significant roles in regulation of plant growth, secondary metabolism, development, defense and response to various abiotic and biotic stresses. Up to now, there is no report on molecular and functional characterization of bZIP transcription factors in Picrorhiza kurrooa, a medicinally important endangered herb of north western Himalayan region. The species is widely used in several commercially available drug formulations like livomap, livocare and livplus for treatment of liver disorders. The medicinal properties of P. kurrooa are due to bioactive picrosides, synthesized by isoprenoid pathway. The promoters of regulatory genes viz., Pkdxs and Pkhmgr of picroside biosynthesis pathway have been reported to contain ACGT cis-acting elements (bzip motifs) at different locations. In this study, the functionality of ACGT motifs was confirmed by electrophoretic mobility shift assay (EMSA). Full length PkbZIP gene was cloned through RACE method. In silico analysis revealed PkbZIP contained a conserved signature bZIP domain (N-x7-R/K-x9-L-x6-L-x6-L). Transcriptional activation and subcellular localization assay confirmed nuclear localization of PkbZIP. EMSA with purified PkbZIP confirmed in vitro binding of PkbZIP to ACGT elements in Pkdxs and Pkhmgr promoters. The expression pattern of PkbZIP positively correlated with the picroside (active medicinal component of the plant) content in different tissues, at 15 °C and under light conditions. Transient overexpression in Nicotiana benthamiana suggested a role of PkbZIP in terpenoid biosynthesis. PkbZIP might prove as potential candidate gene to be used in genetic engineering program for enhancing the picroside content in P. kurrooa.

The Impact Marketing Mix on Indigenous Business Development in Uzbekistan: A Regression Analysis

This study investigates the formulation of a marketing strategy framework specifically designed for small and medium-sized enterprises (SMEs) in Uzbekistan. SMEs have several obstacles in establishing brand awareness, enticing clients, and optimizing financial profits in the current fiercely competitive business landscape. To surmount these challenges and maintain expansion, SMEs can utilize the extended marketing mix approach, which comprises the elements of product, price, place, promotion, people, process, and physical evidence. This study employed a blend of descriptive and exploratory research methodologies, integrating both quantitative data-gathering approaches. A survey was undertaken to assess the perspectives of entrepreneurs about the implementation of marketing mix strategies SMEs. The study examined the influence of the correlation between marketing mix variables on the development of SMEs through regression analysis with the help of STATA software. The results emphasized the significance of integrating marketing mix elements for SMEs to improve their marketing endeavors, foster brand loyalty, and get a competitive edge. This study enhances comprehension of marketing strategies for small and medium-sized firms in developing economies such as Uzbekistan and offers valuable insights for policymakers and entrepreneurs in promoting the expansion of SMEs in the economy.

Hypoxia-induced TRPM7 promotes glycolytic metabolism and progression in hepatocellular carcinoma

BackgroundHypoxia disrupts glucose metabolism in hepatocellular carcinoma (HCC). Transient receptor potential cation channel, subfamily M, member 7 (TRPM7) plays an ontogenetic role. Thus, we aimed to explore the regulation of TRPM7 by hypoxia-induced factor (HIF) and its underlying mechanisms in HCC. Methodshypoxia was induced in multiple HCC cells using 1% O2 or CoCl2 treatment, and subsequently blocked using siRNAs targeting HIF-1α or HIF-2α as well as a HIF-1α protein synthesis inhibitor. The levels of HIF-1α and TRPM7 were assessed using quantitative PCR (qPCR) and Western blot analysis. Chromatin immunoprecipitation (ChIP) and luciferase assays were performed to observe the regulation of TRPM7 promoter regions by HIF-1α. A PCR array was utilized to screen glucose metabolism-related enzymes in HEK293 cells overexpressing TRPM7 induced by tetracycline, and then verified in TRPM7-overexpressed huh7 cells. Finally, CCK-8, transwell, scratch and tumor formation experiments in nude mice were conducted to examine the effect of TRPM7 on proliferation and metastasis in HCC. ResultsExposure to hypoxia led to increase the levels of TRPM7 and HIF-1α in HCC cells, which were inhibited by HIF-1α siRNA or enhanced by HIF-1α overexpression. HIF-1α directly bound to two hypoxia response elements (HREs) in the TRPM7 promoter. Several glycolytic metabolism-related enzymes, were simultaneously upregulated in HEK293 and huh7 cells overexpressing TRPM7 during hypoxia. In vitro and in vivo experiments demonstrated that TRPM7 promoted the proliferation and metastasis of HCC cells. ConclusionsTRPM7 was directly transcriptionally regulated by HIF-1α, leading to glycolytic metabolic reprogramming and the promotion of HCC proliferation and metastasis in vitro and in vivo. Our findings suggest that TRPM7 might be a potential diagnostic indicator and therapeutic target for HCC.

New genes helped acorn barnacles adapt to a sessile lifestyle.

Barnacles are the only sessile lineages among crustaceans, and their sessile life begins with the settlement of swimming larvae (cyprids) and the formation of protective shells. These processes are crucial for adaptation to a sessile lifestyle, but the underlying molecular mechanisms remain poorly understood. While investigating these mechanisms in the acorn barnacle, Amphibalanus amphitrite, we discovered a new gene, bcs-6, which is involved in the energy metabolism of cyprid settlement and originated from a transposon by acquiring the promoter and cis-regulatory element. Unlike mollusks, the barnacle shell comprises alternate layers of chitin and calcite and requires another new gene, bsf, which generates silk-like fibers that efficiently bind chitin and aggregate calcite in the aquatic environment. Our findings highlight the importance of exploring new genes in unique adaptative scenarios, and the results will provide important insights into gene origin and material development.

Positive regulation of the Eucommia rubber biosynthesis-related gene EuFPS1 by EuWRKY30 in Eucommia ulmoides

Eucommia rubber is a secondary metabolite from Eucommia ulmoides that has attracted much attention because of its unique properties and enormous potential for application. However, the transcriptional mechanism regulating its biosynthesis has not yet been determined. Farnesyl pyrophosphate synthase is a key enzyme in the Eucommia rubber biosynthesis. In this study, the promoter of EuFPS1 was used as bait, EuWRKY30 was screened from the cDNA library of EuFPS1 via a yeast one-hybrid system. EuWRKY30 belongs to the WRKY IIa subfamily and contains a WRKY domain and a C2H2 zinc finger motif, and the expressed protein is located in the nucleus. EuWRKY30 and EuFPS1 exhibited similar tissue expression patterns, and yeast one-hybrid and dual-luciferase experiments confirmed that EuWRKY30 directly binds to the W-box element in the EuFPS1 promoter and activates its expression. Moreover, the overexpression of EuWRKY30 significantly upregulated the expression level of EuFPS1, further increasing the density of the rubber particles and Eucommia rubber content. The results of this study indicated that EuWRKY30 positively regulates EuFPS1, which plays a critical role in the synthesis of Eucommia rubber, provided a basis for further analysis of the underlying transcriptional regulatory mechanisms.

Activation of CTU2 expression by LXR promotes the development of hepatocellular carcinoma.

Cytosolic thiouridylase 2 (CTU2) is an enzyme modifying transfer RNAs post-transcriptionally, which has been implicated in breast cancer and melanoma development. And we found CTU2 participated in hepatocellular carcinoma (HCC) progression here. HepG2 cells as well as xenograft nude mice model were employed to investigate the role of CTU2 in HCC development in vitro and in vivo respectively. Further, we defined CTU2 as a Liver X receptor (LXR) targeted gene, with a typical LXR element in the CTU2 promoter. CTU2 expression was activated by LXR agonist and depressed by LXR knockout. Interestingly, we also found CTU2 took part in lipogenesis by directly enhancing the synthesis of lipogenic proteins, which provided a novel mechanism for LXR regulating lipid synthesis. Meanwhile, lipogenesis was active during cell proliferation, particularly in tumor cells. Reduction of CTU2 expression was related to reduced tumor burden and synergized anti-tumor effect of LXR ligands by inducing tumor cell apoptosis and inhibiting cell proliferation. Taken together, our study identified CTU2 as an LXR target gene. Inhibition of CTU2 expression could enhance the anti-tumor effect of LXR ligand in HCC, identifying CTU2 as a promising target for HCC treatment and providing a novel strategy for the application of LXR agonists in anti-tumor effect.

Activation of the native PHYTOENE SYNTHASE 1 promoter by modifying near-miss cis-acting elements induces carotenoid biosynthesis in embryogenic rice callus

Key messageModification of silent latent endosperm-enabled promoters (SLEEPERs) allows the ectopic activation of non-expressed metabolic genes in rice callusMetabolic engineering in plants typically involves transgene expression or the mutation of endogenous genes. An alternative is promoter modification, where small changes in the promoter sequence allow genes to be switched on or off in particular tissues. To activate silent genes in rice endosperm, we screened native promoters for near-miss cis-acting elements that can be converted to endosperm-active regulatory motifs. We chose rice PHYTOENE SYNTHASE 1 (PSY1), encoding the enzyme responsible for the first committed step in the carotenoid biosynthesis pathway, because it is not expressed in rice endosperm. We identified six motifs within a 120-bp region, upstream of the transcriptional start site, which differed from endosperm-active elements by up to four nucleotides. We mutated four motifs to match functional elements in the endosperm-active BCH2 promoter, and this promoter was able to drive GFP expression in callus and in seeds of regenerated plants. The 4 M promoter was not sufficient to drive PSY1 expression, so we mutated the remaining two elements and used the resulting 6 M promoter to drive PSY1 expression in combination with a PDS transgene. This resulted in deep orange callus tissue indicating the accumulation of carotenoids, which was subsequently confirmed by targeted metabolomics analysis. PSY1 expression driven by the uncorrected or 4 M variants of the promoter plus a PDS transgene produced callus that lacked carotenoids. These results confirm that the adjustment of promoter elements can facilitate the ectopic activation of endogenous plant promoters in rice callus and endosperm and most likely in other tissues and plant species.

Functional differentiation and genetic diversity of rice cation exchanger (CAX) genes and their potential use in rice improvement

Cation exchanger (CAX) genes play an important role in plant growth/development and response to biotic and abiotic stresses. Here, we tried to obtain important information on the functionalities and phenotypic effects of CAX gene family by systematic analyses of their expression patterns, genetic diversity (gene CDS haplotypes, structural variations, gene presence/absence variations) in 3010 rice genomes and nine parents of 496 Huanghuazhan introgression lines, the frequency shifts of the predominant gcHaps at these loci to artificial selection during modern breeding, and their association with tolerances to several abiotic stresses. Significant amounts of variation also exist in the cis-regulatory elements (CREs) of the OsCAX gene promoters in 50 high-quality rice genomes. The functional differentiation of OsCAX gene family were reflected primarily by their tissue and development specific expression patterns and in varied responses to different treatments, by unique sets of CREs in their promoters and their associations with specific agronomic traits/abiotic stress tolerances. Our results indicated that OsCAX1a and OsCAX2 as general signal transporters were in many processes of rice growth/development and responses to diverse environments, but they might be of less value in rice improvement. OsCAX1b, OsCAX1c, OsCAX3 and OsCAX4 was expected to be of potential value in rice improvement because of their associations with specific traits, responsiveness to specific abiotic stresses or phytohormones, and relatively high gcHap and CRE diversity. Our strategy was demonstrated to be highly efficient to obtain important genetic information on genes/alleles of specific gene family and can be used to systematically characterize the other rice gene families.

Transcription Factor Binding Site in Promoter Determines the Pattern of Plasmid-Based Transgene Expression In Vivo.

Understanding the regulation of transgene expression is critical for the success of plasmid-based gene therapy and vaccine development. In this study, we used two sets of plasmid vectors containing secreted embryonic alkaline phosphatase or the mouse IL-10 gene as a reporter and investigated the role of promoter elements in regulating transgene expression in vivo. We demonstrated in mice that hydrodynamic transfer of plasmids with the CMV promoter resulted in a high level of reporter gene expression that declined rapidly over time. In contrast, when plasmids with albumin promoters were used, a lower but sustained gene expression pattern was observed. We also found that plasmids containing a shorter CMV promoter sequence with fewer transcription factor binding sites showed a decrease in the peak level of gene expression without changing the overall pattern of reporter gene expression. The replacement of regulatory elements in the CMV promoter with a single regulatory element of the albumin promoter changed the pattern of transient gene expression seen in the CMV promoter to a pattern of sustained gene expression identical to that of a full albumin promoter. ChIP analyses demonstrated an elevated binding of acetylated histones and TATA box-binding protein to the promoter carrying regulatory elements of the albumin promoter. These results suggest that the strength of a promoter is determined by the number of appropriate transcription factor binding sites, while gene expression persistence is determined by the presence of regulatory elements capable of recruiting epigenetic modifying complexes that make the promoter accessible for transcription. This study provides important insights into the mechanisms underlying gene expression regulation in vivo, which can be used to improve plasmid-based gene therapy and vaccine development.