GUS Gene Research Articles

The Competing Endogenous RNAs Regulatory Genes Network Mediates Leaf Shape Variation and Main Effector Gene Function in Mulberry Plant (Morus alba)

Mulberry plants (Morus alba) have leaf shapes, ranging from unlobed to lobed, which are crucial for yield, growth, and adaptability, indicating their ability to adapt to their environment. Competing endogenous RNAs (ceRNAs) constitute a web of RNAs within the organism’s transcriptional regulatory system, including protein-coding genes (mRNAs), microRNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and others. In this study, samples for ceRNA sequencing were categorized into two groups: whole leaves and lobed leaves, each group with three replicates. In addition, we isolated, cloned, and characterized the precursor miRNA (miR156x) from the leaves of M. alba. miR156x precursor had a length of 107 base pairs and a minimum folding free energy of 50.27 kcal/mol. We constructed a pCAMBIA-35S-GUS-miR156x dual overexpression vector and established a transient transformation system for mulberry. At an optimal transformation solution (OD600 = 0.7), the GUS gene showed a higher expression in the leaves of transiently transformed mulberry with miR156x overexpression, four days after transformation, while the target genes of miR156x had decreased expression in the same leaves. Investigations into the transgenic mulberry plants uncovered various modifications to physio-chemical parameters including POD, SOD, PRO, MDA, soluble proteins and sugars, and chlorophyl content. miRNAs in the plants were found to act as negative regulators of gene expression in response to changes in leaf shape regulation, which was confirmed in vitro using dual-luciferase reporter assays. Subsequently, we cloned Maspl3 in vitro and conducted GST-Pull down assays, obtaining multiple proteins that interacted with the Maspl3 gene. This indicates that the miR156x/Maspl3/MSTRG.25812.1 regulatory module contributes to the differences in mulberry leaf shape.

Promoter cloning and activities analysis of JmLFY, a key gene for flowering in Juglans mandshurica.

Juglans mandshurica (Manchurian walnut) is a precious timber and woody grain and oil species in Northeast China. The heterodichogamous characteristic phenomenon resulted in the non-synchronous flowering and development of male and female flowers, which limited the mating and the yield and quality of fruits. LFY is a core gene in the flowering regulatory networks, which has been cloned in J. mandshurica, and the function has also been verified preliminarily. In this study, the JmLFY promoter sequence with different lengths of 5'-deletion (pLFY1-pLFY6) were cloned and conducted bioinformatics analysis, the promoter activities were analyzed by detecting their driving activity to GUS gene in the tobacco plants that transformed with different promoter sequence stably or transiently. After that, the interaction between JmSOC1 and JmLFY gene promoter was also analyzed via yeast single-hybrid. The results showed that the promoter sequence contains core cis-acting elements essential for eukaryotic promoters, hormone response elements, defense- and stress-responsive elements, flowering-related elements, etc. Transgenic tobacco plants with pLFY1 were obtained by Agrobacterium infection using the pCAMBIA1301 expression vector, and the GUS gene driven by the JmLFY promoter was detected to express in the leaf, stem, flower, and root of the transformed tobacco plant, which indicated that the obtained JmLFY promoter had driving activity. GUS histochemical staining and enzyme activity detection showed that promoter fragments with different lengths had promoter activity and could respond to the induction of long photoperiod, low temperature, salicylic acid (SA), IAA, GA3, and methyl jasmonate (MeJA). The core regulatory region of JmLFY gene promoter in J. mandshurica was between -657 bp and -1,904 bp. Point-to-point validation of yeast single-hybrid confirmed the interaction between JmSOC1 and JmLFY gene promoter, which indicated that JmLFY gene is the downstream target of JmSOC1. These results reveal relevant factors affecting JmLFY gene expression and clarify the molecular mechanism of JmLFY gene regulation in the flower developmental partially, which will provide a theoretical basis for regulating the flowering time by regulating JmLFY gene expression in J. mandshurica.

Daptomycin production enhancement by ARTP mutagenesis and fermentation optimization in Streptomyces roseosporus.

We evaluated whether the randomness of mutation breeding can be regulated through a double-reporter system. We hope that by establishing a new precursor feeding strategy, the production capacity of industrial microorganisms after pilot scale-up can be further improved. In this study, the industrial strain Streptomyces roseosporus L2796 was used as the starter strain for daptomycin production, and a double-reporter system with the kanamycin resistance gene Neo and the chromogenic gene gusA was constructed to screen for high-yield strain L2201 through atmospheric and room temperature plasma (ARTP). Furthermore, the composition of the culture medium and the parameters of precursor replenishment were optimized, resulting in a significant enhancement of the daptomycin yield of the mutant strain L2201(752.67mg/l). This study successfully screened a high-yield strain of daptomycin through a double-reporter system combined with ARTP mutation. The expression level of two reporter genes can evaluate the strength of dptEp promoter, which can stimulate the expression level of dptE in the biosynthesis of daptomycin, thus producing more daptomycin. The developed multi-stage feeding rate strategy provides a novel way to increase daptomycin in industrial fermentation.

Aztreonam is a novel chemical inducer that promotes Agrobacteium transformation and lateral root development in soybean.

Agrobacterium-mediated soybean transformation is the simplest method of gene transfer. However, the low transformation due to the intractable nature of soybean genotypes hinders this process. The use of biochemicals (acetosyringone, cinnamic acid, flavonoids, etc.) plays an important role in increasing soybean transformation. These biochemicals induce chemotaxis and virulence gene activation during the infection process. Here we identified a biochemical, aztreonam (a monobactam), for high agrobacterium-mediated transformation in soybean. The soybean explants from three genotypes were inoculated with A. tumefaciens (GV3101) harboring the pMDC32 vector containing hpt or the GmUbi-35S-GUS vector containing the GUS gene during two separate events. High transient GUS expression was obtained during cotyledon explant culture on MS media supplemented with 2.5 mg/L aztreonam. The aztreonam-treated explants showed high efficiency in transient and stable transformation as compared to the untreated control. The transformation of aztreonam-treated explants during seed imbibition resulted in an average of 21.1% as compared to 13.2% in control by using the pMDC32 vector and 28.5 and 20.7% while using the GUS gene cassette, respectively. Based on these findings, the metabolic analysis of the explant after aztreonam treatment was assessed. The high accumulation of flavonoids was identified during an untargeted metabolic analysis. The quantification results showed a significantly high accumulation of the four compounds, i.e., genistein, apigenin, naringenin, and genistin, in cotyledon explants after 18 hours of aztreonam treatment. Alongside this, aztreonam also had some surprising effects on root elongation and lateral root formation when compared to indole-3-butyric acid (IBA). Our findings were limited to soybeans. However, the discovery of aztreonam and its effect on triggering flavonoids could lead to the potential role of aztreonam in the agrobacterium-mediated transformation of different crops.

Assessment of Gus Expression Induced by Anti-Sense OsPPO Gene Promoter and Antioxidant Enzymatic Assays in Response to Drought and Heavy Metal Stress in Transgenic Arabidopsis thaliana

Abiotic stresses, including drought and heavy metals, are detrimental to plant growth and development and enormously reduce agricultural yields. Plants may quickly change their transcriptome in response to various stressful conditions. Plants develop many defense mechanisms to respond to various stresses that can be classified into morphological, physiological, and biochemical responses. Polyphenol oxidases (PPOs) are one of the self-protective enzymes found in plants except for Arabidopsis. Currently, drought and heavy metals were applied exogenously to transgenic A. thaliana lines (transformed with Oryza sativa PPO promoter fused to the GUS reporter gene). The current study mainly focused on the systematic pathway by which plants respond to stressors. The aim of this study is to investigate the effect/expression of PPO and antioxidant defense system against abiotic stresses. A. thaliana was treated with different concentrations of polyethylene glycols. At 30% PEG, maximum fold induction (1.9) was seen after 12 h. Overall, various concentrations (5%, 20%, and 30%) induced PPO expression after 6, 12, and 24 h. Moreover, three different concentrations of Cu (50 µM, 100 µM, 200 µM) and Ni (50 µM, 100 µM, 200 µM) for 6, 12, and 24 h were also applied. It was observed that the expression profiling of the OsPPO promoter induced GUS gene expression in response to Cu and Ni treatments. The maximum fold induction (15.03) of GUS was observed in 100 µM of Cu after 24 h. In the case of Ni, maximum fold induction of (7.78) was observed at 100 µM after 24 h. So, both Cu and Ni showed a similar pattern of induction at 100 µM after 24 h. In conclusion, the efficiency of the PPOGUS promoter can be operated to assess the response of plants to various abiotic stimuli.

Agrobacterium-mediated transformation of Nang Thom Cho Dao, an indica rice variety

Agrobacterium-mediated transformation is the most efficient method to transfer gene of interest into the plant genome. However, the transformation efficiency of this method with indica rice varieties is still low and needs to be improved. In this study, the Agrobacterium-mediated method was used to transfer reporter genes into the Nang Thom Cho Dao, an indica rice variety. Different transformation parameters, such as selection to used hygromycin at concentrations of 0, 25, 30, 40, and 50 mg/L, bacterial density, and infected duration, were tested and optimized. The results showed that 30 mg/L of hygromycin was the most appropriate concentration for selecting transgenic callus in Nang Thom Cho Dao. In addition, the highest transformation efficiency of this Nang Thom Cho Dao rice cultivar was observed at the bacterial suspension density OD600nm of 0.1, the infected duration of 20 minutes, and the 3-day co-culture period. The presence and expression of transgenes were confirmed by gus staining and PCR with specific primers for hptII and gus genes. This transformation procedure should be used for further studies in genetic engineering of Nang Thom Cho Dao and other indica rice varieties.

Identification of the inducible activity in the promoter of the soybean BBI-DII gene exposed to abiotic stress or abscisic acid.

The expression of the soybean Bowman-Birk proteinase isoinhibitor DII (BBI-DII) gene and the inducible activity of its promoter were studied under salt, drought, low temperature, and abscisic acid (ABA) exposure conditions. The BBI-DII gene was induced by salt, drought, low temperature, and ABA, and the relative expression levels were 103.09-, 107.01-, 17.25- and 27.24-fold, respectively, compared with the untreated control. The putative promoter, designated BP1 (- 1255 to + 872bp), located 5'-upstream of the BBI-DII gene was cloned. The expression of the GUS gene in pCAM-BP1 transgenic tobacco plants was highest at 5h after treatment with salt, drought, low temperature and ABA, especially under salt and drought. Using histochemical staining and fluorescence analysis of GUS, BP1 activity under salt and drought conditions after 5h was 1.03 and 1.07-fold, respectively, compared with that of the CaMV35S promoter. Based on a 5' deletion analysis, the segment (+ 41 to + 474bp) was the basal region that responded to salt and drought, whereas the segment (- 820 to + 41bp) was the area that responded to increased salt and drought activity. The BP2 (- 820 to + 872) activities were 0.98- and 1.02-fold compared with that of BP1 under salt and drought conditions and was 435bp shorter than BP1. The salt- and drought-inducible activities of the BP2 promoter in the roots, stems, and leaves of transgenic tobacco plants were stable. Taken together, BP2 is more suitable than the BP1 promoter for the study and molecular breeding of stress-resistant soybean plants.

Agrobacterium-mediated Genetic Transformation of two Peanut (Arachis hypogaea L.) Varieties using Salinity Tolerant PDH45 Gene

An efficient Agrobacterium-mediated genetic transformation protocol has been developed in the present investigation to integrate salinity tolerant gene pea DNA helicase (PDH45) in two peanut varieties, Dhaka-1 and BARI Chinabadam-8. Decapitated half-embryo explants of the above-mentioned varieties were also used to develop a transformation compatible in vitro regeneration system. MS medium supplemented with 5.0 mg/l BAP and 0.5 mg/l Kn showed maximum response for shoot induction in both varieties. Half strength of MS medium supplemented with 0.2 mg/l IBA showed better response towards root induction for both varieties. Genetic transformation experiments were performed using Agrobacterium strain LBA4404 containing pCAMBIA1301-PDH45 conferring reporter gus gene, hygromycin resistance hpt gene, and salinity tolerant gene pdh45. The transformation variables were optimized, OD600 1.4 with 45 minutes of incubation period, and three days of co-culture showed the best performance. The transformed shoots were selected using 30 mg/l hygromycin. The overall transformation frequency was very low, where 0.10% shoots of Dhaka-1 and 0.07% shoots of BARI Chinabadam-8 survived the selection pressure. The stable integration of the transgene in the transformed shoots was confirmed through PCR analysis. Plant Tissue Cult. & Biotech. 33(1): 37-46, 2023 (June)

Establishment of a transformation system in close relatives of wheat under the assistance of TaWOX5

Species closely related to wheat are important genetic resources for agricultural production, functional genomics studies and wheat improvement. In this study, a wheat gene related to regeneration, TaWOX5, was applied to establish the Agrobacterium-mediated transformation systems of Triticum monococcum, hexaploid triticale, and rye (Secale cereale L.) using their immature embryos. Transgenic plants were efficiently generated. During the transformation process, the Agrobacterium infection efficiency was assessed by histochemical staining for β-glucuronidase (GUS). Finally, the transgenic nature of regenerated plants was verified by polymerase chain reaction (PCR)-based genotyping for the presence of the GUS and bialaphos resistance (bar) genes, histochemical staining for GUS protein, and the QuickStix strip assay for bar protein. The transformation efficiency of T. monococcum genotype PI428182 was 94.4%; the efficiencies of four hexaploid triticale genotypes Lin456, ZS3297, ZS1257, and ZS3224 were 52.1, 41.2, 19.4, and 16.0%, respectively; and the transformation efficiency of rye cultivar Lanzhou Heimai was 7.8%. Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) analyses indicated that the GUS transgenes were integrated into the distal or near centromere (proximal) regions of the chromosomes in transgenic T. monococcum and hexaploid triticale plants. In the transgenic hexaploid triticale plants, the foreign DNA fragment was randomly integrated into the AABB and RR genomes. Furthermore, the transgene was almost stably inherited in the next generation by Mendel’s law. The findings in this study will promote the genetic improvement of the three plant species for grain or forage production and the improvement of cereal species including wheat for functional genomics studies.

Cloning and expression analysis of U6 promoters in Panax quinquefolius

The U6 promoter is an important element driving sgRNA transcription in the CRISPR/Cas9 system. Seven PqU6 promo-ter sequences were cloned from the gDNA of Panax quinquefolium, and the transcriptional activation ability of the seven promoters was studied. In this study, seven PqU6 promoter sequences with a length of about 1 300 bp were cloned from the adventitious roots of P. quinquefolium cultivated for 5 weeks. Bioinformatics tools were used to analyze the sequence characteristics of PqU6 promoters, and the fusion expression vectors of GUS gene driven by PqU6-P were constructed. Tobacco leaves were transformed by Agrobacterium tumefaciens-mediated method for activity detection. The seven PqU6 promoters were truncated from the 5'-end to reach 283, 287, 279, 289, 295, 289, and 283 bp, respectively. The vectors for detection of promoter activity were constructed with GUS as a reported gene and used to transform P. quinquefolium callus and tobacco leaves. The results showed that seven PqU6 promoter sequences(PqU6-1P to PqU6-7P) were cloned from the gDNA of P. quinquefolium, with the length ranged from 1 246 bp to 1 308 bp. Sequence comparison results showed that the seven PqU6 promoter sequences and the AtU6-P promoter all had USE and TATA boxes, which are essential elements affecting the transcriptional activity of the U6 promoter. The results of GUS staining and enzyme activity test showed that all the seven PqU6 promoters had transcriptional activity. The PqU6-7P with a length of 1 269 bp had the highest transcriptional activity, 1.31 times that of the positive control P-35S. When the seven PqU6 promoters were truncated from the 5'-end(PqU6-1PA to PqU6-7PA), their transcriptional activities were different in tobacco leaves and P. quinquefolium callus. The transcriptional activity of PqU6-7PA promoter(283 bp) was 1.59 times that of AtU6-P promoter(292 bp) when the recipient material was P. quinquefolium callus. The findings provide more ideal endogenous U6 promoters for CRISPR/Cas9 technology in ginseng and other medicinal plants.

NODULE INCEPTION: a direct regulator of gibberellin biosynthesis during symbiotic nodulation.

This article is a Commentary on Gao et al. (2023), 239: 459–465.

Patients with hereditary angioedema and patients with SARS-CoV-2-associated pneumonia – what can be similar?

IntroductionHereditary angioedema (HAE) is a rare, autosomal dominant genetic disorder that leads to sporadic episodes of swelling, which can affect any part of the body. In most cases, HAE is caused by a deficiency (type I) or dysfunction (type II) of C1-inhibitor (C1-INH), with an estimated prevalence of 1:10.000–1:50.000. HAE type I characterized by low antigenic and functional C1-INH levels, HAE type II characterized by normal or elevated antigenic but low functional C1-INH levels. Materials and MethodsStudy groups included: patients with genetically confirmed HAE type I (group 1, n = 49); patients with genetically confirmed HAE type II (group 2, n = 13); patients with SARS-CoV-2-associated pneumonia with a median recovery period of 5 months (group 3, n = 11); patients with SARS-CoV-2 infection and without pneumonia with a median recovery period of 5 months (group 4, n = 41). The expression of the C1-INH gene was measured when HAE patients were not on prophylactic treatment. The results presented as a median (25%; 75%). C1-INH expression levels (normal range: 1.47 REU (1.07; 2.01)) were measured using real-time PCR and normalized to the level of GUS gene expression. All subjects consented to the study. ResultsThe expression of the C1-INH gene in the first group was 0.31 REU (0.19; 0.60); in the second – 1.45 REU (1.26; 2.87); in the third – 0.47 REU (0.32; 0.88) and in the fourth – 0.76 REU (0.57; 1.29). We revealed that the third group had the similar range of C1-INH gene expression as in patients with HAE type I who have primary quantitative deficiency of C1-INH. Only in 27.3% (3/11) patients from group 3 and in 70.7% (29/41) patients from group 4 after recover from SARS-CoV-2 infection the expression level of the C1-INH gene have returned to the normal range. ConclusionA recent studies displays that SARS-CoV-2 infection in human body directly causes a deficit in C1-INH, also there are evidences of the successful use of replacement therapy with a C1-INH in severe cases of COVID-19. However, the reason for the persistence of an acquired C1-INH deficiency in patients even with a several month recovery period remains to be elucidated.

Cloning and functional analysis of the promoter of a UDP-glycosyltransferase gene from Panax quinquefolium L.

Pq3-O-UGT2 is a key UDP-glycosyltransferase gene in the ginsenoside biosynthesis pathway of Panax quinquefolium L. Functional analysis of promoter sequences plays an important role in understanding the regulation of functional gene expression. In this study, chromosome walking technology was used to isolate the 1399 bp sequence upstream of the ATG initiation codon of Pq3-O-UGT2. Bioinformatics analysis shows that the promoter sequence contains a large number of putative cis-acting elements responsive to exogenous and endogenous factors. The full-length promoter and six 5′ terminal truncations were fused with the GUS reporter gene to test their activities. The results of histochemical staining showed that a strong GUS activity were observed in flowers, siliques, leaves, stems and roots of transgenic Arabidopsis containing the full length Pq3-O-UGT2 promoter. Fluorometric assays showed that the highest enzyme activity is the full-length promoter with 4370 pmol 4-MU/min/mg protein, and the shortest promoter containing P-198::GUS with 45 pmol 4-MU/min/mg protein was sufficient to activate GUS expression. In addition, extended light, low temperatures, MeJA, ABA, NAA and GA3 were selected to investigate the Pq3-O-UGT2 promoter in response to abiotic stress and hormone treatment. The GUS activity of Pq3-O-UGT2 full-length promoter (P-1399) was 2.12 times that of the control plant after MeJA treatment, suggesting that P-1399 is a MeJA-inducible promoter. Furthermore, the assay results showed that ABA could extensively induce GUS expression in five 5′ truncated promoter transgenic plants, except P-198. These experimental results will help us to better explore the regulatory mechanisms in the promoter region of the Pq3-O-UGT2 gene and contribute to further studies of the molecular mechanisms of glycosylation in ginseng plants. The Pq3-O-UGT2 promoter was successfully cloned. Seven 5′ truncations could drive the expression of GUS gene in transgenic Arabidopsis plants and the full-length promoter is a MeJA-inducible promoter.

Genome-Wide Investigation of Apyrase (APY) Genes in Peanut (Arachis hypogaea L.) and Functional Characterization of a Pod-Abundant Expression Promoter AhAPY2-1p

Peanut (Arachis hypogaea L.) is an important food and feed crop worldwide and is affected by various biotic and abiotic stresses. The cellular ATP levels decrease significantly during stress as ATP molecules move to extracellular spaces, resulting in increased ROS production and cell apoptosis. Apyrases (APYs) are the nucleoside phosphatase (NPTs) superfamily members and play an important role in regulating cellular ATP levels under stress. We identified 17 APY homologs in A. hypogaea (AhAPYs), and their phylogenetic relationships, conserved motifs, putative miRNAs targeting different AhAPYs, cis-regulatory elements, etc., were studied in detail. The transcriptome expression data were used to observe the expression patterns in different tissues and under stress conditions. We found that the AhAPY2-1 gene showed abundant expression in the pericarp. As the pericarp is a key defense organ against environmental stress and promoters are the key elements regulating gene expression, we functionally characterized the AhAPY2-1 promoter for its possible use in future breeding programs. The functional characterization of AhAPY2-1P in transgenic Arabidopsis plants showed that it effectively regulated GUS gene expression in the pericarp. GUS expression was also detected in flowers of transgenic Arabidopsis plants. Overall, these results strongly suggest that APYs are an important future research subject for peanut and other crops, and AhPAY2-1P can be used to drive the resistance-related genes in a pericarp-specific manner to enhance the defensive abilities of the pericarp.

Combination of Hairy Root and Whole-Plant Transformation Protocols to Achieve Efficient CRISPR/Cas9 Genome Editing in Soybean

The new gene-editing technology CRISPR/Cas system has been widely used for genome engineering in various organisms. Since the CRISPR/Cas gene-editing system has a certain possibility of low efficiency and the whole plant transformation of soybean is time-consuming and laborious, it is important to evaluate the editing efficiency of designed CRISPR constructs before the stable whole plant transformation process starts. Here, we provide a modified protocol for generating transgenic hairy soybean roots to assess the efficiency of guide RNA (gRNA) sequences of the CRISPR/Cas constructs within 14 days. The cost- and space-effective protocol was first tested in transgenic soybean harboring the GUS reporter gene for the efficiency of different gRNA sequences. Targeted DNA mutations were detected in 71.43-97.62% of the transgenic hairy roots analyzed as evident by GUS staining and DNA sequencing of the target region. Among the four designed gene-editing sites, the highest editing efficiency occurred at the 3' terminal of the GUS gene. In addition to the reporter gene, the protocol was tested for the gene-editing of 26 soybean genes. Among the gRNAs selected for stable transformation, the editing efficiency of hairy root transformation and stable transformation ranged from 5% to 88.8% and 2.7% to 80%, respectively. The editing efficiencies of stable transformation were positively correlated with those of hairy root transformation with a Pearson correlation coefficient (r) of 0.83. Our results demonstrated that soybean hairy root transformation could rapidly assess the efficiency of designed gRNA sequences on genome editing. This method can not only be directly applied to the functional study of root-specific genes, but more importantly, it can be applied to the pre-screening of gRNA in CRISPR/Cas gene editing.

Application of Nicotinamide to Culture Medium Improves the Efficiency of Genome Editing in Hexaploid Wheat.

Histone acetylation is the earliest and most well-characterized of post-translation modifications. It is mediated by histone acetyltransferases (HAT) and histone deacetylases (HDAC). Histone acetylation could change the chromatin structure and status and further regulate gene transcription. In this study, nicotinamide, a histone deacetylase inhibitor (HDACi), was used to enhance the efficiency of gene editing in wheat. Transgenic immature and mature wheat embryos harboring a non-mutated GUS gene, the Cas9 and a GUS-targeting sgRNA were treated with nicotinamide in two concentrations (2.5 and 5 mM) for 2, 7, and 14 days in comparison with a no-treatment control. The nicotinamide treatment resulted in GUS mutations in up to 36% of regenerated plants, whereas no mutants were obtained from the non-treated embryos. The highest efficiency was achieved when treated with 2.5 mM nicotinamide for 14 days. To further validate the impact of nicotinamide treatment on the effectiveness of genome editing, the endogenous TaWaxy gene, which is responsible for amylose synthesis, was tested. Utilizing the aforementioned nicotinamide concentration to treat embryos containing the molecular components for editing the TaWaxy gene, the editing efficiency could be increased to 30.3% and 13.3%, respectively, for immature and mature embryos in comparison to the 0% efficiency observed in the control group. In addition, nicotinamide treatment during transformation progress could also improve the efficiency of genome editing approximately threefold in a base editing experiment. Nicotinamide, as a novel approach, may be employed to improve the editing efficacy of low-efficiency genome editing tools such as base editing and prime editing (PE) systems in wheat.

Protocol for efficient ginseng transformation

Panax ginseng is a major medicinal crop with pharmaceutical efficacy derived from ginsenoside metabolites. Despite its genome information, the inefficiency of ginseng transformation hinders the study of the molecular mechanism of ginseng plant metabolism. Thus, this protocol aimed to develop an easy and efficient system for ginseng transformation. We established a transformation system using ginseng callus cultured in a liquid medium, which has a higher rate compared with cotyledon explant. In addition, Agrobacterium tumefaciens has been used for plant transformation. Compared with the LBA4404 strain, C58C1 was inappropriate for ginseng transformation using ginseng callus. We induced and maintained calli in liquid medium and cut them into small pieces before infection. After infection, we selected calli that survived from the selection media until identification of newly growing cells. In β-glucuronidase (GUS) assay, the expression of the GUS gene was observed in cells that were newly generated from explants. We treated calli with 0.05 M of MgSO4 before infection. After MgSO4 pre-treatment, the transformation efficiency of growing cells around infected callus was increased than non-treated control. Moreover, we constructed and introduced a visible reporter RUBY system to easily identify transformed cells. Using this system, we could identify the cells by a red color with naked eyes. Based on our transformation protocol, the success rate has increased to 77.27% in surviving lines during selection culture. This stable ginseng transformation could facilitate the overexpression and knockout of ginseng lines for functional or synthetic biological studies. We re-designed the ginseng transformation protocol using ginseng calli for explant material, and MgSO4 pretreatment was conducted before Agrobacterium infection. RUBY reporter was successfully introduced in ginseng.

Chlormequat chloride treatment inhibits grapevine stem growth via the VviRAP2.12 - VviEXPA7 regulatory module

Treatment with chlormequat chloride (CCC) can lead to dwarfism, which has been studied in many plants. However, the underlying molecular mechanisms have been less studied. Herein, the phenotypic changes and gene expression levels of grapevine in response to CCC were investigated. Results showed that the height of grape plants became significantly shorter after CCC treatment, especially between the first two stems above the roots. Transcriptome sequencing was then performed and analyzed, 120 differentially expressed genes (DEGs) were detected in the stems after CCC treatment compared to the control. The gene ontology (GO) enrichment results showed that processes such as cellulose synthase (UDP-forming) activity, regulation of the auxin-mediated signaling pathway, and the cellulose biosynthetic process were common. In addition, weighted gene co-expression network analysis (WGCNA) identified the strong correlation between the MEbrown module and stem development. VviEXAP7 was selected from the genes belonging to key GO pathways and WGCNA modules. The quantitative real-time PCR (qRT-PCR) results showed that VviEXPA7 was significantly downregulated because of the CCC treatment. Additionally, analysis of VviEXPA7 promoter activity showed that VviEXPA7 activated GUS gene expression, and was induced by CCC, abscisic acid (ABA), and other hormones. VviEXPA7 was used as a target gene for transcription factor prediction, and after employing the PlantTFDB and JASPAR databases, VviRAP2.12 was identified as a transcription factor that might regulate VviEXPA7. qRT-PCR revealed that VviRAP2.12 was significantly upregulated after CCC treatment. Finally, the interaction between VviEXPA7 and VviRAP2.12 was verified by the dual-luciferase assay. Thus, the current study shows that CCC decreased the expression of the cell growth-related gene VviEXPA7 by promoting the expression of VviRAP2.12, which resulted in dwarfed plants. This study generated new information that will increase our understanding of the mechanism of action of CCC and provides a scientific basis for simplified cultivation of grapes.

Molecular cloning and functional characterization of the promoter of a novel Aspergillus flavus inducible gene (AhOMT1) from peanut.

Peanut is an important oil and food legume crop grown in more than one hundred countries, but the yield and quality are often impaired by different pathogens and diseases, especially aflatoxins jeopardizing human health and causing global concerns. For better management of aflatoxin contamination, we report the cloning and characterization of a novel A. flavus inducible promoter of the O-methyltransferase gene (AhOMT1) from peanut. The AhOMT1 gene was identified as the highest inducible gene by A. flavus infection through genome-wide microarray analysis and verified by qRT-PCR analysis. AhOMT1 gene was studied in detail, and its promoter, fussed with the GUS gene, was introduced into Arabidopsis to generate homozygous transgenic lines. Expression of GUS gene was studied in transgenic plants under the infection of A. flavus. The analysis of AhOMT1 gene characterized by in silico assay, RNAseq, and qRT-PCR revealed minute expression in different organs and tissues with trace or no response to low temperature, drought, hormones, Ca2+, and bacterial stresses, but highly induced by A. flavus infection. It contains four exons encoding 297 aa predicted to transfer the methyl group of S-adenosyl-L-methionine (SAM). The promoter contains different cis-elements responsible for its expression characteristics. Functional characterization of AhOMT1P in transgenic Arabidopsis plants demonstrated highly inducible behavior only under A. flavus infection. The transgenic plants did not show GUS expression in any tissue(s) without inoculation of A. flavus spores. However, GUS activity increased significantly after inoculation of A. flavus and maintained a high level of expression after 48 hours of infection. These results provided a novel way for future management of peanut aflatoxins contamination through driving resistance genes in A. flavus inducible manner.

Bacillus subtilis small replicative plasmid collection incorporating five distinct origins of replication and compatible with Golden Gate DNA assembly.

In order to facilitate Golden Gate DNA assembly, we have constructed a collection of Bacillus subtilis replicative plasmids representing five origins of replication derived from plasmids pUB110, pE194, pWV01, pBS72, and pTH1030. The first three of these plasmids use rolling circle replication and the latter two use theta replication. All of the plasmids carry the same multiple cloning site surrounded by transcriptional terminators. The plasmids are about three kilobases in size, allowing them to be easily amplified by inverse PCR using a common set of primers to generate cloning-ready amplicons. This plasmid PCR amplification approach also facilitates a workflow that eliminates Escherichia coli as a shuttle intermediate. All of the plasmids lack a site for at least three of the type IIS restriction enzymes BbsI, BsaI, Esp3I, PaqCI, or SapI, making them compatible with Golden Gate DNA assembly. We have demonstrated the utility of the plasmids by performing Golden Gate assembly of gusA and bgaB-reporter gene fragments and in expressing plasmid-borne red fluorescent protein under the control of RNA polymerase from bacteriophage K1E.