Cloning and functional analysis of flavanone 3-hydroxylase gene in Rhododendron hybridum Hort / 生物工程学报

Flavanone 3-hydroxylase (F3H) is a key enzyme in the synthesis of phycocyanidins. In this experiment, the petals of red Rhododendron hybridum Hort. at different developmental stages were used as experimental materials. The R. hybridum flavanone 3-hydroxylase (RhF3H) gene was cloned using reverse transcription PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques, and bioinformatics analyses were performed. Petal RhF3H gene expression at different developmental stages were analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR). A pET-28a-RhF3H prokaryotic expression vector was constructed for the preparation and purification of RhF3H protein. A pCAMBIA1302-RhF3H overexpression vector was constructed for genetic transformation in Arabidopsis thaliana by Agrobacterium-mediated method. The results showed that the R. hybridum Hort. RhF3H gene is 1 245 bp long, with an open reading frame of 1 092 bp, encoding 363 amino acids. It contains a Fe2+ binding motif and a 2-ketoglutarate binding motif of the dioxygenase superfamily. Phylogenetic analysis showed that the R. hybridum RhF3H protein is most closely related to the Vaccinium corymbosum F3H protein. qRT-PCR analysis showed that the expression level of the red R. hybridum RhF3H gene tended to increase and then decrease in the petals at different developmental stages, with the highest expression at middle opening stage. The results of the prokaryotic expression showed that the size of the induced protein of the constructed prokaryotic expression vector pET-28a-RhF3H was about 40 kDa, which was similar to the theoretical value. Transgenic RhF3H Arabidopsis thaliana plants were successfully obtained, and PCR identification and β-glucuronidase (GUS) staining demonstrated that the RhF3H gene was integrated into the genome of A. thaliana plants. qRT-PCR, total flavonoid and anthocyanin contentanalysis showed that RhF3H was significantly higher expressed in the transgenic A. thaliana relative to that of the wild type, and its total flavonoid and anthocyanin content were significantly increased. This study provides a theoretical basis for investigating the function of RhF3H gene, as well as for studying the molecular mechanism of flower color in R. simsiib Planch.

Effects of plant growth-promoting rhizobacteria Klebsiella michiganensis TS8 and Lelliottia jeotgali MR2 on the growth and cadmium uptake of Arabidopsis thaliana under cadmium stress / 生物工程学报

In this study, the effects of two plant growth-promoting bacteria Klebsiella michiganensis TS8 and Lelliottia Jeotgali MR2 on the growth and cadmium (Cd) uptake of Arabidopsis thaliana under Cd stress were explored. A wild-type Arabidopsis thaliana was selected as the experimental plant and was planted at different Cd concentrations. MR2 and TS8 bacterial suspensions were sprayed onto the rhizospheric soil during the planting process. The initial Cd concentration of the bought soil was 14.17 mg/kg, which was used as the pot soil of the low-concentration Cd treatment group (LC). The concentration of soil Cd at high-concentration Cd treatment group (HC) were 200 mg/kg higher than that at LC group. Compared with the control group, MR2 suspension significantly promoted the growth of A. thaliana at both low and high concentrations, while TS8 strain and MR2_TS8 mixture only exhibited growth-promoting effect at high concentration. However, it was noteworthy that, TS8 suspension significantly reduced the Cd content in the underground parts of A. thaliana (60% and 59%), and significantly improved the Cd content in the aboveground parts of A. thaliana (234% and 35%) at both low and high concentrations. In addition, at low concentration, both single strain and mixed strains significantly improved the transformation from reducible Cd to acid-extractable Cd in soil, promoted Cd intake, and thereby reduced the total Cd content in soil. Therefore, the rational application of plant growth-promoting bacteria may improve crop yield and remediate Cd contamination in soil.

Ginsenosides in

Ginsenosides are a series of glycosylated triterpenoids which belong to protopanaxadiol (PPD)-, protopanaxatriol (PPT)-, ocotillol (OCT)- and oleanane (OA)-type saponins known as active compounds of

Cloning and Characterization of 5-enopy ruvylshikimate-3-phosphate Synthase from Fragaria vesca

Abstract To discover and isolate a glyphosate-resistant gene from Fragaria vesca through gene mining. An open reading frame (ORF) of 1563 bp encoding EPSPSwas amplified from Fragaria vesca (FvEPSPS). FvEPSPS (Genebank: XP004306932.1) encodes a polypeptide of 520 amino acids and it has hightly homologous with EPSPS from other plants. qRT-PCR analysis showed that the FvEPSPS was expressed extensively in all tissues including leaves, roots and stems, with higher expression in leaves. Furthermore, transgenic Arabidopsis Thaliana exhibited 10 mM glyphosate to resistance. Therefore, this research offers a new glyphosate-resistant gene for development of transgenic crops.

Molecular cloning and functional characterization of CmFT (FLOWERING LOCUS T) from Cucumis melo L.

CmFT homologous gene in muskmelon was obtained by homologous cloning, introducing CmFT gene by Agrobacteriummediated transformation. The results of subcellular localization showed that CmFT protein was expressed in cytoplasm and nucleus. qRTPCR results showed that the expression levels of AtLFY, AtFT, AtCO, AtFLC, AtSOC1 and AtAP1 were upregulated in the 35S::MeFT Arabidopsis line. The CmFT gene was introduced into wild-type Arabidopsis by Agrobacterium-mediated transformation, and the growth status of T2 transgenic Arabidopsis thaliana and wild-type A. thaliana was observed. The results showed that wild-type Arabidopsis began to bolt on the 25th day after sowing, we can initially confirm that the FT gene of melon can promote the early flowering of melon in the growth and development of melon.

Prokaryotic expression, purification and functional identification of epidermal pattern factors in Arabidopsis thaliana / 生物工程学报

Stomatal density is important for crop yield. In this paper, we studied the epidermal pattern factors (EPFs) related to stomatal development. Prokaryotic expression vectors were constructed to obtain EPFs. Then the relationship between EPFs and hydrogen sulfide (H2S) was established. First, AtEPF1, AtEPF2 and AtEPFL9 were cloned and constructed to pET28a vectors. Then recombinant plasmids pET28a-AtEPF1, pET28a-AtEPF2 and pET28a-AtEPFL9 were digested and sequenced, showing successful construction. Finally, they were transformed into E. coli BL21(DE3) separately and induced to express by isopropyl β-D-galactoside (IPTG). The optimized expression conditions including IPTG concentration (0.5, 0.3 and 0.05 mmol/L), temperature (28 °C, 28 °C and 16 °C) and induction time (16 h, 16 h and 20 h) were obtained. The bands of purified proteins were about 18 kDa, 19 kDa and 14.5 kDa, respectively. In order to identify their function, the purified AtEPF2 and AtEPFL9 were presented to Arabidopsis thaliana seedlings. Interestingly, the H2S production rate decreased or increased compared with the control, showing significant differences. That is, EPFs affected the production of endogenous H2S in plants. These results provide a foundation for further study of the relationship between H2S and EPFs on stomatal development, but also a possible way to increase the yield or enhance the stress resistance.

Arabidopsis SIMILAR TO RCD-ONE genes are ubiquitous and respond to multiple abiotic stresses through diverse signaling pathways

The SIMILAR TO RCD-ONE (SROs) have been characterized as a group of plant-specific proteins which play importantfunctions in stress responses and development. Here, we analyze the expression profiles of six SRO genes under differentstress treatments in Arabidopsis. Our results revealed that RCD1 play an essential role in plant responses to variousenvironmental stresses. SRO1 has partially overlapping functions with RCD1 in plant response to HgCl2 and H2O2 stress.Analysis of the transcriptional expression of SROs indicated that both of the RCD1 and SRO1 transcripts were up-regulatedby HgCl2 and light, not by other stresses, and that of SRO5 was induced by salt. Expression of SRO3 and SRO4 were notinfluenced by stresses. The different effects of these stresses on the expression of the SRO genes indicate that the SROfamily is regulated by multiple signaling pathways. Sequence analyses of the SRO proteins implicate a highly preservedprotein structure and are specific to plants, which might have implications for functional conservation. The ubiquitousexpression and nuclear localization of SRO family suggested that their function might be related to transcription factorregulation and complex formation. Taken together, SRO family is critical for proper plant development and multiple stressresponses.

Cloning and molecular characterization of a putative habanero pepper SERK1 cDNA expressed during somatic and zygotic embryogenesis

Background: Plant gene homologs that control cell differentiation can be used as biotechnological tools to study the in vitro cell proliferation competence of tissue culture-recalcitrant species such as peppers. It has been demonstrated that SERK1 homologs enhance embryogenic competence when overexpressed in transformed tissues; therefore, cloning of a pepper SERK1 homolog was performed to further evaluate its biotechnological potential. Results: A Capsicum chinense SERK full-length cDNA (CchSERK1) was cloned and characterized at the molecular level. Its deduced amino acid sequence exhibits high identity with sequences annotated as SERK1 and predicted-SERK2 homologs in the genomes of the Capsicum annuum CM-334 and Zunla-1 varieties, respectively, and with SERK1 homologs from members of the Solanaceae family. Transcription of CchSERK1 in plant tissues, measured by quantitative RT-PCR, was higher in stems, flowers, and roots but lower in leaves and floral primordia. During seed development, CchSERK1 was transcribed in all zygotic stages, with higher expression at 14 days post anthesis. During somatic embryogenesis, CchSERK1 was transcribed at all differentiation stages, with a high increment in the heart stage and lower levels at the torpedo/cotyledonal stages. Conclusion: DNA sequence alignments and gene expression patterns suggest that CchSERK1 is the C. chinense SERK1 homolog. Significant levels of CchSERK1 transcripts were found in tissues with cell differentiation activities such as vascular axes and during the development of zygotic and somatic embryos. These results suggest that CchSERK1 might have regulatory functions in cell differentiation and could be used as a biotechnological tool to study the recalcitrance of peppers to proliferate in vitro.

Carbohydrates and polyphenolics of extracts from genetically altered plant acts as catalysts for in vitro synthesis of silver nanoparticle

Eco-friendly biosynthetic approach for silver nanoparticles production using plant extracts is an exciting advancement inbio-nanotechnology and has been successfully attempted in nearly 41 plant species. However, an established model plantsystem for systematically unraveling the biochemical components required for silver nanoparticles production is lacking.Here we used Arabidopsis thaliana as the model plant for silver nanoparticles biosynthesis in vitro. Employing biochemical, spectroscopic methods, selected mutants and over-expressor plants of Arabidopsis involved in pleotropicfunctions and sugar homeostasis, we show that carbohydrates, polyphenolics and glyco-proteins are essential componentswhich stimulated silver nanoparticles synthesis. Using molecular genetics as a tool, our data enforces the requirement ofsugar conjugated proteins as essentials for AgNPs synthesis over protein alone. Additionally, a comparative analysis ofAgNPs synthesis using the aqueous extracts of some of the plant species found in a brackish water ecosystem (Gracilaria,Potamogeton, Enteromorpha and Scendesmus) were explored. Plant extract of Potamogeton showed the highest potentialof nanoparticles production comparable to that of Arabidopsis among the species tested. Silver nanoparticles production inthe model plant Arabidopsis not only opens up a possibility of using molecular genetics tool to understand the biochemicalpathways and components in detail for its synthesis.

AtMBD4: A methylated DNA binding protein negatively regulates a subset of phosphate starvation genes

DNA methylation is an important epigenetic modification that governs transcriptional regulation. The methylation mark isread by a special class of proteins called methyl-CpG-binding domain proteins. The role of DNA methylation has beenfound in X-chromosome inactivation, genomic imprinting, transposon silencing, and self-incompatibility. Recently,remodeling of global DNA methylation was demonstrated in Arabidopsis during low phosphate availability. The presentstudy reports that AtMBD4 gene of Arabidopsis negatively regulates phosphate starvation. The T-DNA insertion mutation atthe AtMBD4 locus exhibited altered root architecture as compared to wild-type plants. Using microarray hybridization andanalysis, an increased transcript accumulation of 242 genes was observed in the mutant. Many of these genes were relatedto phosphate transporters and transcription factors, involved in phosphate starvation response. Comparison of data ofatmbd4 mutant with publicly available microarray data of phosphate starvation response indicated the role of AtMBD4protein in phosphate starvation response. Further, promoter analysis of up-regulated genes suggested that cis-regulatoryelements like MBS, W-box, and B1BS are more prominent in the promoters of up-regulated genes. Upon performing amethylation-specific PCR, a decreased DNA methylation in the promoter regions of up-regulated genes was observed. Theaccumulation of anthocyanin and inorganic phosphate in the atmbd4 mutant was found to be higher than the wild-typeplant. Altered root morphology, up-regulation of phosphate starvation-induced genes in atmbd4 mutant suggests thatAtMBD4 negatively regulates the phosphate starvation response.

Exploring Genetic Variability among Mutant Accessions of Arabidopsis thaliana L.

Genetic diversity among 12 accessions of Arabidopsis thaliana possessing Pi transporter genes (Pht), compared to the related wild type, have been investigated using inter-simple sequence repeats (ISSR) and two biochemical marker systems; sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and isozymes. ISSR fingerprints clearly distinguished the 13 accessions and differentiate between the wild type and its mutant accessions. The six primers revealed a total of 79 fragments, ranging from 5 to 23 bands per primer (average 13.16 bands per primer). The polymorphism ranged between 44% for (GA)8C primer and 100% for the other primers. The cluster of ISSR patterns reflected high polymorphism (90.7%). AG repeat was the most common among the targeted units because it represented by the highest number of bands, whereas, AC and GA repeats gave the lowest number. No distinct polymorphism was observed in isozyme and SDS-PAGE patterns where ACP produced only one monomorphic band. Biochemical results could not characterize any variable behavior for mutant accessions compared to wild type even in the presence of three different members of Pht1 genes. In conclusion, ISSR markers are recommended for exploring genetic variability among different Arabidopsis mutants.

Expression analysis of the Arabidopsis thaliana AtSpen2 gene, and its relationship with other plant genes encoding Spen proteins

Abstract Proteins of the Split ends (Spen) family are characterized by an N-terminal domain, with one or more RNA recognition motifs and a SPOC domain. In Arabidopsis thaliana, the Spen protein FPA is involved in the control of flowering time as a component of an autonomous pathway independent of photoperiod. The A. thaliana genome encodes another gene for a putative Spen protein at the locus At4g12640, herein named AtSpen2. Bioinformatics analysis of the AtSPEN2 SPOC domain revealed low sequence similarity with the FPA SPOC domain, which was markedly lower than that found in other Spen proteins from unrelated plant species. To provide experimental information about the function of AtSpen2, A. thaliana plants were transformed with gene constructs of its promoter region with uidA::gfp reporter genes; the expression was observed in vascular tissues of leaves and roots, as well as in ovules and developing embryos. There was absence of a notable phenotype in knockout and overexpressing lines, suggesting that its function in plants might be specific to certain endogenous or environmental conditions. Our results suggest that the function of Atspen2 diverged from that of fpa due in part to their different transcription expression pattern and divergence of the regulatory SPOC domain.

Study on expression of metallothionein 2 gene in safflower / 中草药

Objective: To obtain the transgenic safflower plants which expressed Arabidopsis thaliana metallothionein 2 (MT2) gene, and lay a foundation for development of MT products. Methods: The oleosin-MT gene was obtained from pEASY-oleosin-MT by Nco I/Hind III, then was inserted into plant expression vector pOP. The recombinant plasmid named pOP-oleosin-MT was transferred into Agrobacterium tumefaciens EHA105.The oleosin-MT gene was introduced into safflowers via Agrobacterium-mediated method and positive transgenic plants were determined by PCR analysis. Results: The recombinant plasmid pOP-oleosin-MT was successfully constructed. PCR and Southern blotting analysis confirmed that MT gene was integrated into the genome of safflower plant and three transgenic plants were obtained. Conclusion: The safflower regeneration system is constructed successfully and MT gene is successfully transformed into safflower plant.

Construction of expression vector of PgMYB4 gene in Panax ginseng and analysis on drought resisting / 中草药

Objective: To construct the expression vector of PgMYB4 gene in Panax ginseng and study its function of the drought resisting in Arabidopsis thaliana. Methods: A P. ginseng gene PgMYB4 was cloned by RT-PCR analysis, further, recombinant plasmid vector with PgMYB4 was transformed into wild-type plants of A. thaliana by Agrobacterium tumefacies-mediated Floral Dip method. Transgenic A. thaliana with the expression of PgMYB4 was obtained, further compared with wild-type plants of A. thaliana, their determination of physiologic index related to drought stress was detected. Results: The cDNA (named a PgMYB4) contained a 735 bp open reading frame, encoded 245 amino acids and the predicted molecular weight was 27.914 KDa; Under the condition of drought stress, the growth of transgenic A. thaliana was obviously better than wild-type A. thaliana. Compared with wild-type A. thaliana, the decreased range of relative chlorophyll content in the leaves of transgenic plants of A. thaliana was smaller and the proline content of transgenic plants of A. thaliana increased significantly. The water loss of transgenic plants of A. thaliana was less than that of Wild-type transgenic plants of A. thaliana. Conclusion: Ginseng PgMYB4 gene has the ability of resistance to drought stress.

Construction of CHI gene expression vector of safflower and its transformation in Arabidopsis thaliana / 中草药

Objective: Plant expression vector of chalcone isomerase in safflower was built and its function was verified by over- expression of CHI in Arabidopsis thaliana. Methods: CHI isolated from safflower in our previous study was introduced by BamH I and EcoR I restriction sites and constructed into the over-expression vector pBASTA-CHI containing 35S promoter, transformed into A. thaliana by Flora-dip method. T2 plants of transgenic A. thaliana were detected by PCR and content of total flavones. Results: Plant expression vector containing the safflower CHI gene was built and over expressed in A. thaliana successfully. T2 plants of transgenic A. thaliana were obtained. Conclusion: PCR of transgenic T2 in A. thaliana is detected that CHI gene of safflower has been integrated into the A. thaliana genome, flavone content is determined in leaves, and the results show that the flavone in transgenetic A. thaliana is higher than that in wild type, the highest strain increases by nearly 2.3 times.

Arabidopsis thaliana as Bioindicator of Fungal VOCs in Indoor Air

In this paper, we demonstrate the ability of Arabidopsis thaliana to detect different mixtures of volatile organic compounds (VOCs) emitted by the common indoor fungus, Aspergillus versicolor, and demonstrate the potential usage of the plant as a bioindicator to monitor fungal VOCs in indoor air. We evaluated the volatile production of Aspergillus versicolor strains SRRC 108 (NRRL 3449) and SRRC 2559 (ATCC 32662) grown on nutrient rich fungal medium, and grown under conditions to mimic the substrate encountered in the built environment where fungi would typically grow indoors (moist wallboard and ceiling tiles). Using headspace solid phase microextraction/gas chromatography-mass spectrometry, we analyzed VOC profiles of the two strains. The most abundant compound produced by both strains on all three media was 1-octen-3-ol. Strain SRRC 2559 made several terpenes not detected from strain SRRC 108. Using a split-plate bioassay, we grew Arabidopsis thaliana in a shared atmosphere with VOCs from the two strains of Aspergillus versicolor grown on yeast extract sucrose medium. The VOCs emitted by SRRC 2559 had an adverse impact on seed germination and plant growth. Chemical standards of individual VOCs from the Aspergillus versicolor mixture (2-methyl-1-butanol, 3-methyl-1-butanol, 1-octen-3-ol, limonene, and β-farnesene), and β-caryophyllene were tested one by one in seed germination and vegetative plant growth assays. The most inhibitory compound to both seed germination and plant growth was 1-octen-3-ol. Our data suggest that Arabidopsis is a useful model for monitoring indoor air quality as it is sensitive to naturally emitted fungal volatile mixtures as well as to chemical standards of individual compounds, and it exhibits relatively quick concentration- and duration-dependent responses.

Expression, purification and characterization of arabinose-5-phosphate isomerase from Arabidopsis thaliana / 生物工程学报

Arabinose-5-phosphate isomerase (KdsD) is the first key limiting enzyme in the biosynthesis of 3-deoxy-D-manno-octulosonate (KDO). KdsD gene was cloned into prokaryotic expression vector pET-HTT by seamless DNA cloning method and the amount of soluble recombinant protein was expressed in a soluble form in E. coli BL21 (DE3) after induction of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The target protein was separated and purified by Ni-NTA affinity chromatography and size exclusion chromatography, and its purity was more than 85%. Size exclusion chromatography showed that KdsD protein existed in three forms: polymers, dimmers, and monomers in water solution, different from microbial KdsD enzyme with the four polymers in water solution. Further, the purified protein was identified through Western blotting and MALDI-TOF MASS technology. The results of activity assay showed that the optimum pH and temperature of AtKdsD isomerase activities were 8.0 and 37 ℃, respectively. The enzyme was activated by metal protease inhibitor EDTA (5 mmol/L) and inhibited by some metal ions at lower concentration, especially with Co²⁺ and Cd²⁺ metal ion. Furthermore, when D-arabinose-5-phosphate (A5P) was used as substrate, Km and Vmax of AtKdsD values were 0.16 mmol/L, 0.18 mmol/L·min. The affinity of AtKdsD was higher than KdsD in E. coli combined with substrate. Above results have laid a foundation for the KdsD protein structure and function for its potential industrial application.

Identification of plant defence regulators through transcriptional profiling of Arabidopsis thaliana cdd1 mutant.

A sustainable balance between defence and growth is essential for optimal fitness under pathogen stress. Plants activate immune response at the cost of normal metabolic requirements. Thus, plants that constitutively activate defence are deprived of growth. Arabidopsis thaliana mutant constitutive defence without defect in growth and development1 (cdd1) is an exception. The cdd1 mutant is constitutive for salicylic acid accumulation, signalling, and defence against biotrophic and hemibiotrophic pathogens, without having much impact on growth. Thus, cdd1 offers an ideal genetic background to identify novel regulators of plant defence. Here we report the differential gene expression profile between cdd1 and wild-type plants as obtained by microarray hybridization. Expression of several defence-related genes also supports constitutive activation of defence in cdd1. We screened T-DNA insertion mutant lines of selected genes, for resistance against virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Through bacterial resistance, callose deposition and pathogenesis-associated expression analyses, we identified four novel regulators of plant defence. Resistance levels in the mutants suggest that At2g19810 and [rom] At5g05790 are positive regulators, whereas At1g61370 and At3g42790 are negative regulators of plant defence against bacterial pathogens.

Phenotypic plasticity and ecotypic variations in growth and flowering time of Arabidopsis thaliana (L.) under different light and temperature conditions.

Four ecotypes of A. thaliana (L.) (Ct-1, Pf-0, Old-1 and Per-1) from low to high latitudes were grown under different light (300 mmol photon m-2s-1 and 150 mmol photon m-2s-1) and temperature (22 and 14 ºC) conditions to investigate their effects on phenotypic plasticity and ecotypic variations in plant growth and first flowering time. The results suggest that in A. thaliana low temperature decreases both phenotypic plasticity and ecotypic variations in first flowering time and total dry matter at final harvest under different light intensities. Relative growth rate is the most stable parameter of A. thaliana that is hardly affected by ecotype (no effect), light (no effect) or temperature (small effect) and this may one of the reason why A. thaliana is widely distributed on earth as a result of adaptations to different environments.

Hpa1 harpin needs nitroxyl terminus to promote vegetative growth and leaf photosynthesis in Arabidopsis.

Hpa1 is a harpin protein produced by Xanthomonas oryzae, an important bacterial pathogen of rice, and has the growth-promoting activity in plants. To understand the molecular basis for the function of Hpa1, we generated an inactive variant protein, Hpa1ΔNT, by deleting the nitroxyl-terminal region of the Hpa1 sequence and compared Hpa1ΔNT with the full-length protein in terms of the effects on vegetative growth and related physiological responses in Arabidopsis. When Hpa1 was applied to plants, it acted to enhance the vegetative growth but did not affect the floral development. Enhanced plant growth was accompanied by induced expression of growth-promoting genes in plant leaves. The growth-promoting activity of Hpa1 was further correlated with a physiological consequence shown as promoted leaf photosynthesis as a result of facilitated CO2 conduction through leaf stomata and mesophyll cells. On the contrary, plant growth, growth-promoting gene expression, and the physiological consequence changed little in response to the Hpa1ΔNT treatment. These analyses suggest that Hpa1 requires the nitroxyl-terminus to facilitate CO2 transport inside leaf cells and promote leaf photosynthesis and vegetative growth of the plant.