Effect of Calmodulin-like Gene (CML) Overexpression on Stilbene Biosynthesis in Cell Cultures of Vitis amurensis Rupr.

Stilbenes are plant phenolics known to rapidly accumulate in grapevine and other plants in response to injury or pathogen attack and to exhibit a great variety of healing beneficial effects. It has previously been shown that several calmodulin-like protein (CML) genes were highly up-regulated in cell cultures of wild-growing grapevine Vitis amurensis Rupr. in response to stilbene-modulating conditions, such as stress hormones, UV-C, and stilbene precursors. Both CML functions and stilbene biosynthesis regulation are still poorly understood. In this study, we investigated the effect of overexpression of five VaCML genes on stilbene and biomass accumulation in the transformed cell cultures of V. amurensis. We obtained 16 transgenic cell lines transformed with the VaCML52, VaCML65, VaCML86, VaCML93, and VaCML95 genes (3–4 independent lines per gene) under the control of the double CaMV 35S promoter. HPLC-MS analysis showed that overexpression of the VaCML65 led to a considerable and consistent increase in the content of stilbenes of 3.8–23.7 times in all transformed lines in comparison with the control calli, while biomass accumulation was not affected. Transformation of the V. amurensis cells with other analyzed VaCML genes did not lead to a consistent and considerable effect on stilbene biosynthesis in the cell lines. The results indicate that the VaCML65 gene is implicated in the signaling pathway regulating stilbene biosynthesis as a strong positive regulator and can be useful in viticulture and winemaking for obtaining grape cultivars with a high content of stilbenes and stress resistance.

Guard-Cell-Specific Expression of Phototropin2 C-Terminal Fragment Enhances Leaf Transpiration

Phototropins (phot1 and phot2) are plant-specific blue light receptors that mediate chloroplast movement, stomatal opening, and phototropism. Phototropin is composed of the N-terminus LOV1 and LOV2 domains and the C-terminus Ser/Thr kinase domain. In previous studies, 35-P2CG transgenic plants expressing the phot2 C-terminal fragment–GFP fusion protein (P2CG) under the control of 35S promoter showed constitutive phot2 responses, including chloroplast avoidance response, stomatal opening, and reduced hypocotyl phototropism regardless of blue light, and some detrimental growth phenotypes. In this study, to exclude the detrimental growth phenotypes caused by the ectopic expression of P2C and to improve leaf transpiration, we used the PHOT2 promoter for the endogenous expression of GFP-fused P2C (GP2C) (P2-GP2C) and the BLUS1 promoter for the guard-cell-specific expression of GP2C (B1-GP2C), respectively. In P2-GP2C plants, GP2C expression induced constitutive phototropin responses and a relatively dwarf phenotype as in 35-P2CG plants. In contrast, B1-GP2C plants showed the guard-cell-specific P2C expression that induced constitutive stomatal opening with normal phototropism, chloroplast movement, and growth phenotype. Interestingly, leaf transpiration was significantly improved in B1-GP2C plants compared to that in P2-GP2C plants and WT. Taken together, this transgenic approach could be applied to improve leaf transpiration in indoor plants.

Expression of Phytase gene in transgenic maize with seed-specific promoter 27-kDa γ Zein and constitutive promoter CaMV 35S

Phytase enzymes are applied to animal feed to help animals absorb more nutrients. The use of feed raw materials containing phytase enzymes is expected to reduce the cost of animal feed production. Efforts to increase the phytase content in maize were carried out by improving genetics, in the way of assembling transgenic plants containing high phytase content. The 27-kDa γ Zein promoter is a specific promoter that expresses genes in caryopsis, and promoter CaMV 35S is a constitutive promoter that controls gene expression in all tissues and generally does not depend on the growth phase. Transgenic maize was transformed using Agrobacterium tumefacien infection method on maize B104. The reverse transcriptase polymerase chain reaction (RT-PCR) approach was used to examine the expression of phytase genes in leaves, roots, and caryopsis was done 10, 20, and 30 days after pollination (DAP). The phytase enzyme activity test was also carried out by using the colorimetric phosphomolybdate analysis method to see the phytase enzyme activity in unit µg-1. The results showed that the phytase gene in transgenic plants with the 27-kDa γ Zein promoter was highly expressed in maize caryopsis, but in line Z6.10 was also expressed in leaves, while in the CaMV 35S promoter the phytase gene was only expressed on the leaves. Phytase enzyme activity showed that transgenic maize was higher than non-transgenic maize.

Expression of Penaeidins 3a in Transgenic Duckweed Confers Antibacterial Ability

Background With the development of aquaculture, fish and shrimp diseases have been paid more and more attention in the world. How to improve the immunity of aquatic animals was an urgent problem to be solved. Duckweed (Lemnacecae), as a eukaryote, could be an ideal feedstock for the production of antimicrobial peptides. Result Penaeidins 3a (Pen 3a) from Litopenaeus vannamei was expressed under the control of CaMV-35S promoter in duckweed, Lemna turionifera 5511. Bacteriostatic test by Pen3a duckweed extract showed the antibacterial activity against Escherichia coli and Staphylococcus aureus. Transcriptome analysis of WT and Pen3a duckweed showed different results, and the protein metabolic process was the most up-regulated DEGs. In Pen 3a transgenic duckweed, the expression of sphingolipid metabolism and phagocytosis process-related genes have been significantly up-regulated. Quantitative proteomics suggested a remarkable difference in protein enrichment in metabolic pathways. Conclusion Our study provide a novel solution on aquaculture and water purification. The Pen 3a transgenic duckweed extraction inhibit the growth of gram-negative bacteria, gram-positive bacteria, which could be applied to control the bacteria in lake. The results could lay the foundation for the subsequent production of antibiotics.

Bottleneck Removal of Paclitaxel Biosynthetic Pathway By Overexpression of DBTNBT Gene Under Methyl- β- Cyclodextrin and Coronatine Elicitation in Taxus Baccata L.

Paclitaxel is a highly functionalized diterpenoid that is broadly used for the treatment of several cancer types. This valuable specialized metabolite naturally exists in the inner bark of Taxus species in low amounts. The limited-scale production of paclitaxel in Taxus cell cultures has necessitated the use of several elicitors. Recently, methyl-β-cyclodextrin (CD) and coronatine (COR) have been considered to be highly effective elicitors in producing plant specialized metabolites. Given the limited production of paclitaxel due to the rate limiting enzymes' function, bottleneck removal is conducive to the production of more significant amounts of paclitaxel. In the present study, the full length of DBTNBT coding sequence (CDS), as one of the paclitaxel pathway bottlenecks, was integrated downstream of the CaMV 35S promoter (pCAMBIA1304-DBTNBT) and transiently expressed in Taxus baccata leaves via Agrobacterium tumefaciens and vacuum infiltration method. Paclitaxel production and the expression level of several involved genes were evaluated through different treatments. The transient overexpression of the DBTNBT gene, associated with dual elicitation, resulted in 7.4-fold more paclitaxel production compared with the no-inoculation/no-elicitation control. These ratios were 2.1 and 1.8 in the CD+COR and pCAM treatments, respectively. Among T13αH, T14βH, DBAT, BAPT, DBTNBT, and ABC genes, the most increased expression level belonged to the DBTNBT gene, followed by ABC and BAPT genes. It seems as though in the near future, bottleneck removal could be used on a large scale in Taxus metabolic engineering, resulting in the relative removal of some other bottlenecks and an increase in the final paclitaxel production.

Plant-derived insulator-like sequences for control of transgene expression

Stable and consistent transgene expression is necessary to advance plant biotechnology. Stable expression can be achieved by incorporating enhancer-blocking insulators, which are cisregulatory elements that reduce enhancer interference in gene expression, into transgene constructs. Sufficient insulators for plant use are not available, and their discovery has remained elusive. In this work, we computationally mined the compact genome of Utricularia gibba for insulator sequences and identified short (<1 kb) sequences with potential insulator activity. Based on in vivo tests, three of these effectively mitigate the ectopic transgene expression caused by the Cauliflower Mosaic Virus 35S promoter and do so better than previously reported plant insulators. However, all sequences with apparent insulator activity also decrease the effectiveness of the CaMV 35S promoter, and thus may be more accurately classified as silencers. However, since the insulator effect is proportionately much higher than the silencing effect, these sequences are still useful for plant transformation.

Mastrevirus Rep and RepA Proteins Suppress de novo Transcriptional Gene Silencing

Transcriptional gene silencing (TGS) in plants is a defense mechanism against DNA virus infection. The genomes of viruses in the Geminiviridae family encode several TGS suppressors. In this study, we induced de novo TGS against the transgenic GFP gene encoding green fluorescent protein by expressing a hairpin-shaped self-complementary RNA corresponding to the enhancer region of the 35S promoter (hpE35S). In addition, we examined the TGS suppression activity of proteins encoded in the genome of Tobacco yellow dwarf virus (TYDV, genus Mastrevirus). The results show that the replication-associated protein (Rep) and RepA encoded by TYDV have TGS suppressor activity and lead to decreased accumulation of 24-nt siRNAs. These results suggest that Rep and RepA can block the steps before the loading of siRNAs into Argonaute (AGO) proteins. This is the first report of TGS suppressors in the genus Mastrevirus.

QUALITY OF HELIANTHUS ANNUUS HONEY OBTAINED IN THE CONDITIONS OF RADIOACTIVE CONTAMINATION

Natural honey is a source of vital amino acids, easily digestible carbohydrates, macro, microelements, biologically active substances that determine nutritional, antibacterial and antioxidant properties. In the conditions of man-caused pollution of Polissya of Ukraine due to the accident at the Chernobyl nuclear power plant, systematic control of the quality and safety of beekeeping products is important. To conduct such research, we created a group of twelve bee families - analogs of the Ukrainian breed, medium strength. Families were kept in unified multifunctional hives. At the beginning of the honey harvest, the bee families were transported to the sunflower fields, where they stayed during the blossoming of the plants. The density of radioactive contamination of 137Cs soils where sunflower was grown was 47.0 kBq / m2. We used organoleptic, physicochemical, microscopic, microbiological, and radiological methods in the study. According to standard methods, we studied the species composition of pollen grains, physicochemical parameters of centrifugal, honeycomb, and «zabrus» sunflower honey.(zabrus honey was obtained from wax caps, which we cut with an apiary knife from honeycombs filled with nectar and sealed by bees). The content of lead (Pb) in honey from sunflower obtained in the conditions of Polissya is 1.8 - 2.1 times higher than the State sanitary norms. The largest amount of it is in the centrifugal honey. In acceptable amounts, the heavy metals cadmium (Cd), arsenic (As), and 137Cs were present in honey. Pesticides, dichlorodiphenyltrichloromethylmethane, and hexachlorane were not detected in the samples. We investigated the bactericidal action against bacterial growth of typical cultures of Proteus vulgaris, Escherichia coli, Klebsiella pneumonia, Salmonella Typhimurium, and Staphylococcus aureus. Zubrus sunflower honey showed the highest antimicrobial and antioxidant properties. We found that the value of antioxidant activity (AOA) of sunflower honey depends on the method of its production, duration of storage, and solutions of extracts (alcohol, aqueous) used in research. Laboratory control of transgenic organisms in flowers and sunflower pollen did not reveal the target sequences of the cauliflower mosaic virus (CaMV) 35S promoter and the NOS terminator (nopaline synthase) of the plasmid Agrobacterium tumefaciens.

Phylogenetic and structural analysis of annexins in pea (Pisum sativum L.) and their role in legume-rhizobial symbiosis development

Annexins as Ca2+/phospholipid-binding proteins are involved in the control of many biological processes essential for plant growth and development. In a previous study, we had shown, using a proteomic approach, that the synthesis of two annexins is induced in pea roots in response to rhizobial inoculation. In this study, phylogenetic analysis identified these annexins as PsAnn4 and PsAnn8 based on their homology with annexins from other legumes. The modeling approach allowed us to estimate the structural features of these annexins that might influence their functional activity. To verify the functions of these annexins, we performed comparative proteomic analysis, experiments with calcium influx inhibitors, and localization of labeled proteins. Essential down-regulation of PsAnn4 synthesis in a non-nodulating pea mutant P56 (sym10) suggests an involvement of this annexin in the rhizobial symbiosis. Quantitative RT-PCR analysis showed that PsAnn4 was upregulated at the early stages of symbiosis development, starting from 1–3 days after inoculation to up to 5 days after inoculation, while experiments with the Ca2+ channel blocker LaCl3 revealed its negative influence on this expression. To follow the PsAnn4 protein localization in plant cells, it was fused to the fluorophores such as red fluorescent protein (RFP) and yellow fluorescent protein (YFP) and expressed under the transcriptional regulation of the 35S promoter in Nicotiana benthamiana leaves by infiltration with Agrobacterium tumefaciens. The localization of PsAnn4 in the cell wall or plasma membrane of plant cells may indicate its participation in membrane modification or ion transport. Our results suggest that PsAnn4 may play an important role during the early stages of pea-rhizobial symbiosis development.

Functional Analysis of a Viral Promoter From the Strawberry Vein Banding Virus (SVBV) Chinese Isolate

BackgroundPromoter is an important factor during gene expression in cells. In this study, we cloned a full-length promoter from the strawberry vein banding virus (SVBV) Chinese isolate and produced several its deletion mutants.MethodsThe full-length promoter of SVBV (SP1) and its three deletion mutants (SP2, SP3, and SP4) were amplified using polymerase chain reaction (PCR). The expression activities controlled by the SVBV SP1, SP2, SP3, and SP4 were evaluated using β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes.ResultsOur transient expression assays showed that the SVBV SP1 promoter as well as its three deletion mutants all expressed the reporter genes, but to very different levels. Interestingly, the expression activity driven by the SP1 promoter was much higher than that shown by the CaMV 35S promoter. After stable transformation of a GUS gene into Nicotiana tabacum plants, the transgene expression level driven by the SVBV SP1 promoter was about 2.6-fold greater than that driven by the CaMV 35S promoter. In addition, the GUS gene expression levels could be enhanced by co-infiltrating the plants with the SP1 promoter-driven vector carrying the GUS gene and the vector expressing the SVBV ORF V or ORF VI.ConclusionsThe SVBV Chinese isolate promoter SP1 is a stronger promoter than the CaMV 35S and FLt-US promoter, may be more useful for production of stable transgenic plants.