Association between the allele compositions of major plant developmental genes and frost tolerance in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) germplasm of different origin

Changing climatic conditions with warming winters and shifts in the frequencies of drought, intense rainfall and cold spells together with associated changes in the geographical distribution of arable crops increase the challenges for selecting new varieties. In this context, we aim to contribute to a better understanding of the determinants of barley (Hordeum vulgare) frost tolerance (FRT) and consequent improvements to marker-assisted selection (MAS). Freezing injury in a diversity panel of 121 barley genotypes with different growth habits and origins was assessed using phenotyping based on chlorophyll fluorescence (F_v/F_m) measurements to screen genetic diversity in plants at an early growth stage. The haplotypes of vernalisation and photoperiod genes were determined with PCR, and correlation analyses were done using data from 12 laboratory and field-laboratory FRT tests. Previous results of allelic combinations of VRN-H1/VRN-H2 for FRT were confirmed with these experiments using a larger set of genotypes. The predictive power of polymorphisms in VRN-H1 intron 1 region for FRT was significantly higher than that of the VRN-H1 promoter polymorphism. The vrn-H1/vrn-H2 facultative genotypes had similar or higher FRT than vrn-H1/Vrn-H2 winter genotypes under suboptimal hardening conditions. Genes regulating long-day and short-day photoperiodic responses were significantly correlated with FRT. The most parsimonious model for prediction of FRT was based on polymorphisms in the VRN-H1 intron 1 region, VRN-H2 and PPD-H2 and explained 69% of the variation in FRT.     

Production of proinsulin in marker-free transgenic tobacco plants using <Emphasis Type="Italic">CRE</Emphasis>/loxP system

The demand for INSULIN is increasing rapidly along with the increased number of diabetic patients. Using the CRE/loxP system, we developed a selective marker-free system without crossing to produce PROINSULIN in transgenic plant. In frame of this approach, the induced promoter pRD29A was isolated from Arabidopsis. The CRE recombinase gene was placed under the control of pRD29A between two loxP recombination sites together with the selective NPTII gene. Furthermore, the binary vector with CRE recombinase and PROINSULIN was constructed and introduced into tobacco (Nicotiana tabacum L.) by Agrobacterium-mediated transformation. Gene excision was used to remove the sequence between the two loxP sites at the presence of 200 mM NaCl. PCR analysis showed that self-excision occurred in several T₀ transgenic plants. Transgenic plants without any marker gene successfully expressed PROINSULIN. This auto-excision strategy provides efficient means of removing the selectable marker gene from transgenic plants. It is an efficient method for producing bio-safe recombinant protein and other valuable substances in plants.     

The pharmaceutics from the foreign empire: the molecular pharming of the prokaryotic staphylokinase in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

Here, we present the application of microbiology and biotechnology for the production of recombinant pharmaceutical proteins in plant cells. To the best of our knowledge and belief it is one of few examples of the expression of the prokaryotic staphylokinase (SAK) in the eukaryotic system. Despite the tremendous progress made in the plant biotechnology, most of the heterologous proteins still accumulate to low concentrations in plant tissues. Therefore, the composition of expression cassettes to assure economically feasible level of protein production in plants remains crucial. The aim of our research was obtaining a high concentration of the bacterial anticoagulant factor—staphylokinase, in Arabidopsis thaliana seeds. The coding sequence of staphylokinase was placed under control of the β-phaseolin promoter and cloned between the signal sequence of the seed storage protein 2S2 and the carboxy-terminal KDEL signal sequence. The engineered binary vector pATAG-sak was introduced into Arabidopsis thaliana plants via Agrobacterium tumefaciens-mediated transformation. Analysis of the subsequent generations of Arabidopsis seeds revealed both presence of the sak and nptII transgenes, and the SAK protein. Moreover, a plasminogen activator activity of staphylokinase was observed in the protein extracts from seeds, while such a reaction was not observed in the leaf extracts showing seed-specific activity of the β-phaseolin promoter.     

Regulatory <Emphasis Type="Italic">cis</Emphasis>-elements on citrus peel-specific expressed gene, <Emphasis Type="Italic">CuCRTISO-like</Emphasis>, promoter respond to hormones and abiotic stresses in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

We identified a peel-specific expressed gene in Citrus unshiu fruits by differentially expressed gene (DEG) analysis, which showed a homology with carotenoid isomerase-like genes identified from other plants and, therefore, designated as CuCRTISO-like. Here we determined the promoter sequence of CuCRTISO-like and analyzed histochemical GUS activity using transgenic Arabidopsis plants harboring CuCRTISO-like promoter-GUS gene constructs (pCRTL-Prom1~pCRTL-Prom5 lines). The promoter activity of CuCRTISO-like was detected in the cotyledon at 5 and 10 days after germination (DAG), young leaf, and anther, but not in the cotyledon at 15 DAG and mature leaf. Several cis-acting elements involved in hormones and abiotic stresses are located on the CuCRTISO-like promoter. Salicylic acid and ethylene treatments induced the GUS activity in the pCRTL-prom1 and pCRTL-Prom4 line, respectively. Treatment of drought and wounding stress induced the GUS activity in the pCRTL-Prom4 and pCRTL-Prom3 line, respectively. Heat stress treatment induced GUS activity more strongly as the promoter length decreased except for no GUS activity in the pCRTL-Prom5 line. The CuCRTISO-like expression during fruit maturation of C. unshiu showed a peel-specific expression pattern. Our results suggest that CuCRTISO-like promoter activity is regulated in a developmental and organ-specific manner, and responds to hormones and abiotic stresses.     

Genome-wide comparative analysis of <Emphasis Type="Italic">LEAFY</Emphasis> promoter sequence in angiosperms

Regulation of the flowering mechanism is influenced by many environmental factors. Dissecting the regulatory processes upstream of the LFY (LEAFY) gene will help us to understand the molecular mechanisms of floral induction. In total, 53 LFY sequences were identified in 37 species. Among the 53 selected LFY promoters and after eliminating the short sequences, 47 LFY promoters were analyzed. Comparative genome studies for LFY promoters among plants showed that TATA-box existed in all herbaceous plants. The 1345-bp promoter sequence upstream to hickory LFY gene was cloned and analyzed, together with functional studies. The result of sequence alignment showed that the region of the hickory LFY promoter has only two conserved auxin response elements (AuxRE), whereas other plants had four. The positions of AuxRE in hickory and walnut were the same, but they were different from the positions from other plants. Furthermore the sequence analysis showed that the promoter have TATA-box and CAAT-box motifs. Deletion analysis of these motifs did not block β-glucuronidase (GUS) activity during the transient expression assay, suggesting that it may be a TATA-less promoter. Low temperature and light significantly induced the full-length promoter to increase about two folds of the GUS enzymatic activity, suggesting these environmental factors induced flowering in hickory.     

Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.     

Change in linker DNA conformation upon histone H1.5 binding to nucleosome: Fluorescent microscopy of single complexes

A method for fluorescently labeled DNA synthesis, which makes it possible to assemble mononucleosomes with 40 bp linkers, was developed. Cy3 and Cy5 labels were introduced into the linkers at distances of 10 bp before the first nucleotide and 15 bp after the last nucleotide of the nucleosome positioning DNA sequence, respectively. Without histone H1.5, f luorescence microscopy of single complexes revealed two equally probable states of nucleosomes. The states were different in the linker conformation: the open one with the energy transfer efficiency (E) between the labels of 0.06 and the closed one with E = 0.37, when the linkers are brought together. Binding of histone H1.5 with nucleosomes occurs in a range of nanomolar concentrations, and the complex formation rate is significantly higher as compared with its dissociation rate. The significant convergence of the DNA linkers (E = 0.73) is observed in these complexes together with the higher conformation uniformity in the region where the labels are localized. The developed nucleosomal constructs represent highly sensitive f luorescent sensors that can be used for the analysis of structural linker rearrangements. Also, in combination with microscopy of single complexes, they are suitable for studying the structure of complexes of nucleosomes with different chromatin architectural proteins.     

Identification and Mutational Analysis of <Emphasis Type="Italic">Escherichia coli</Emphasis> Sorbitol-Enhanced Glucose-Repressed <Emphasis Type="Italic">srlA</Emphasis> Promoter Expressed in LB Medium by Using Homologous Recombination and One-Round PCR Products

Escherichia coli has been used for recombinant protein production for many years. However, no native E. coli promoters have been found for constitutive expression in LB medium. To obtain high-expression E. coli promoters active in LB medium, we inserted various promoter regions upstream of eEmRFP that encodes a red fluorescent protein. Among the selected promoters, only colonies of srlA promoter transformants turned red on LB plate. srlA is a gene that regulates sorbitol utilization. The addition of sorbitol enhanced eEmRFP expression but glucose and other sugars repressed, indicating that srlAp is a sorbitol-enhanced glucose-repressed promoter. To analyze the srlAp sequence, a novel site-directed mutagenesis method was developed. Since we demonstrated that homologous recombination in E. coli could occur between 12-bp sequences, 12-bp overlapping sequences were attached to the set of primers that were designed to produce a full-length plasmid, denoted “one-round PCR product.” Using this method, we identified that the srlA promoter region was 100 bp. Further, the sequence adjacent to the start codon was found to be essential for high expression, suggesting that the traditionally used restriction enzyme sites for cloning in the promoter region have hindered expression. The srlA-driven expression system and DNA manipulation with one-round PCR products are useful tools in E. coli genetic engineering.     

Model-based reconstruction of synthetic promoter library in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Objective

To develop an efficient synthetic promoter library for fine-tuned expression of target genes in Corynebacterium glutamicum.

Results

A synthetic promoter library for C. glutamicum was developed based on conserved sequences of the ??10 and ??35 regions. The synthetic promoter library covered a wide range of strengths, ranging from 1 to 193% of the tac promoter. 68 promoters were selected and sequenced for correlation analysis between promoter sequence and strength with a statistical model. A new promoter library was further reconstructed with improved promoter strength and coverage based on the results of correlation analysis. Tandem promoter P70 was finally constructed with increased strength by 121% over the tac promoter. The promoter library developed in this study showed a great potential for applications in metabolic engineering and synthetic biology for the optimization of metabolic networks.

Conclusions

To the best of our knowledge, this is the first reconstruction of synthetic promoter library based on statistical analysis of C. glutamicum.

     

A SNP in the promoter region of the<Emphasis Type="Italic">VvmybA1</Emphasis> gene is responsible for differences in grape berry color between two related bud sports of grape

The VvmybA1 gene in grape (Vitis vinifera) plays a key role in the biosynthesis of anthocyanin. The grape cultivars, ‘Benitaka’ (red color) and ‘Brazil’ (black color) were the result of a bud mutation. ‘Benetika’ was derived from ‘Italia’ (green color) and ‘Brazil’ was developed from ‘Benetika’. Single sequence repeat (SSR) molecular marker analysis was performed in order to demonstrate that the three cultivars have a common pedigree. A sequence analysis of the promoter region and coding sequence of VvmybA1 revealed a base substitution between ‘Benitaka’ and ‘Brazil’ in the promoter region and a deletion of a large DNA fragment in the promoter region of ‘Italia’. Anthocyanin content and expression of the VvmybA1 and UFGT genes in ‘Brazil’ were higher than in ‘Benitaka’ and barely detectable in ‘Italia’. A transient expression system was used to introduce VvmybA1 driven by the three different promoters present in ‘Italia’, ‘Brazil’, and ‘Benitaka’ into somatic embryos of ‘Centennial Seedless’ (Vitis vinifera L.) by Agrobacterium-mediated transformation. This resulted in the production of red cells in the embryos transformed with the constructs of VvmybA1 from ‘Brazil’ and ‘Benitaka’ and no color production in embryos transformed with the VvmybA1 construct from ‘Italia’. In addition, the embryos transformed with the ‘Brazil’ construct had more red color than the embryos transformed with the ‘Benitaka’ construct. These results suggested that a SNP mutation in the promoter region of VvmybA1 in ‘Benitaka’ (red color) was responsible for the color change displayed by ‘Brazil’ (black color).