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.

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.

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

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.

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.

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.

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.

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.

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.

Effect of cryopreservation on the efficiency of exogenous gene, genetic transformation and expression level of Arabidopsis thaliana

Background: Cryopreservation refers to the storage of a living organism at ultra-low-temperature for long-term preservation of plant germplasm. The effect of cryopreservation on the efficiency of exogenous gene genetic transformation and expression level were studied herein. In this work, transgenic Arabidopsis thaliana were successfully conserved in vitro by cryopreservation methods. Results: The effects of osmotic stress due to cryoprotectants during pretreatment and of storage at -196ºC on the stability, the efficiency of genetic transformation and the expression level of exogenous gene were analyzed in Arabidopsis. The results showed that there had not any significant increasing in the efficiency of genetic transformation after cryopreservation, and our observation was not in agreement with earlier reports. Transgenic Arabidopsis lines over-expressing ATOST1 gene were used for the real-time PCR analysis, and the result indicated that the expression of the ATOST1 gene was up-regulated about 2.4-fold in the transgenic seedlings tissues retrieved from cryopreservation than those non-cryopreserved counterparts. Conclusions： Cryopreservation could improve the expression of exogenous gene, however, could not promote the genetic transformation obviously.

Changes in leaf proteome profile of Arabidopsis thaliana in response to salicylic acid.

Salicylic acid (SA) has been implicated in determining the outcome of interactions between many plants and their pathogens. Global changes in response to this phytohormone have been observed at the transcript level, but little is known of how it induces changes in protein abundance. To this end we have investigated the effect of 1 mM SA on soluble proteins of Arabidopsis thaliana leaves by proteomic analysis. An initial study at transcript level has been performed on temporal landscape, which revealed that induction of most of the SA-responsive genes occurs within 3 to 6 h post treatment (HPT) and the expression peaked within 24 HPT. Two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF MS/MS analysis has been used to identify differentially expressed proteins and 63 spots have been identified successfully. This comparative proteomic profiling of SA treated leaves versus control leaves demonstrated the changes of many defence related proteins like pathogenesis related protein 10a (PR10a), diseaseresistance- like protein, putative late blight-resistance protein, WRKY4, MYB4, etc. along with gross increase in the rate of energy production, while other general metabolism rate is slightly toned down, presumably signifying a transition from ‘normal mode’ to ‘defence mode’.

Antimicrobial effects of plant defence peptides expressed by bovine endothelial cells on intracellular pathogens

Background: The actions of plant antimicrobial peptides (PAP) against intracellular pathogens are poorly known. It has been reported that wheat puroindolines show antibacterial activity against Staphylococcus epidermidis endocyted by macrophages. In this work, we evaluated the intracellular antimicrobial activity of PAP gamma-thionin and thionin Thi2.1 produced by bovine endothelial cells against intracellular Staphylococcus aureus and Candida albicans. We used three host-pathogen models: 1) bovine mammary epithelial cells (BMEC)-S. aureus, 2) bovine endothelial cells (BEC)-S. aureus and 3) BEC-C. albicans, and evaluated the effect of conditioned media from BEC producers of PAP (gamma-thionin and thionin Thi2.1). Results: In the first model, conditioned medium (CM) containing Thi2.1 completely inhibited S. aureus intracellular after 24 hrs treatment. In the second model, CM from BEC containing gamma-thionin has a better effect killing intracellular S. aureus for 12-24 hrs incubations than CM from endothelial cells producers of Thi2.1; this was related with an increase of nitric oxide production (~2 times) in BEC infected and treated for 12 hrs with CM containing gamma-thionin, which negatively correlates with bacterial viability. In the third model, CM containing Thi2.1 showed a more potent intracellular fungicidal activity (~85 percent of inhibition) at 24 hrs treatment than CM containing gamma-thionin (~35 percent of inhibition). Conclusions: This work shows new effects of PAP to control intracellular bacterial or fungal infections.

Mutations in retrotransposon AtCOPIA4 compromises resistance to Hyaloperonospora parasitica in Arabidopsis thaliana

Retrotransposons (RTEs) are a principal component of most eukaryotic genomes, representing 50 percent-80 percent of some grass genomes. RTE sequences have been shown to be preferentially present in disease resistance gene clusters in plants. Arabidopsis thaliana has over 1,600 annotated RTE sequences and 56 of these appear to be expressed because of the exact expressed sequence tag (EST) matches and the presence of intact open reading frames. Of the 22 represented in the Affymetrix ATH1 array, AtCOPIA4 was found to be expressed at a higher level than all other RTEs across different developmental stages. Since AtCOPIA4 is located in the RPP5 gene cluster and is adjacent to RPP4 which confers resistance to the downy mildew oomycete Hyaloperonospora parasitica isolate EMWA1, we evaluated AtCOPIA4 mutants for resistance to this pathogen. T-DNA insertional and antisense knockout of AtCOPIA4 was found to reduce the resistance of wild type plants by 2-4 folds. Our results suggest that retrotransposon can be exapted to participate in plant defense response.

Obtainment and Characterization of The Evolved Enzymes From Arabidopsis thaliana Glutathione S-Transferase Zeta Class / 生物化学与生物物理进展

The Arabidopsis thaliana glutathione S-transferases zeta class (AtGSTZ) is a multi-functional enzyme, which plays important role in cellular metabolism and environmental purification. Error-prone PCR and cycles of DNA shuffling were used to construct a mutagenesis library of AtGSTZ. The screening of the resultant libraries was carried out by a pH indicator dye-based colorimetric assay. Nine mutants which enhanced the dichloroacetic acid dechlorination activity were obtained. Among them, NN23 contained 25 amino acid substitutions with the activity improving 120%, whereas NN20 contained 24 amino acid substitutions with the activity improving 102%. EC1 contained 2 amino acid substitutions with the activity improving 47%. The rest 6 mutants contained one amino acid substitution with their activity increasing from 9% to 60%. The enzymatic characterization showed that all the evolved enzymes increased their catalytic efficiencies towards dichloroacetic acid and binding affinity towards glutathione whereas some of them increased the renaturability. However there is no obvious change in their thermostability. Based on these data, functional residues related to catalysis and refolding of AtGSTZ were discussed.

Genetic regulation of flower development.

Flower development provides a model system to study mechanisms that govern pattern formation in plants. Most flowers consist of four organ types that are present in a specific order from the periphery to the centre of the flower. Reviewed here are studies on flower development in two model species: Arabidopsis thaliana and Antirrhinum majus that focus on the molecular genetic analysis of homeotic mutations affecting pattern formation in the flower. Based on these studies a model was proposed that explains how three classes of regulatory genes can together control the development of the correct pattern of organs in the flower. The universality of the basic tenets of the model is apparent fromthe analysis of the homologues of the Arabidopsis genes from other plant species.

Sequencing of ribosomal 18S rRNA gene from Panax pseudoginseng var.notoginseng / 中草药

Object To identify the characteristics of 18S rRNA from the root of Panax pseudoginsengWall var. notoginseng (Burkill) Hoo et Tseng (PGSN). Methods The primers were designed according to the gene sequence of 18S rRNA from the model plant arabidopsis thaliana. 18S rRNA gene sequence of PGSN were cloned and sequenced and compared with that of the model plant A. thaliana and P. pseudoginseng subsp.Wall himalacus var. angustifolius. Results Part of the characteristics of ribosomal 18S rRNA gene sequence of PGSN from Jingxi, Guangxi Province were identified, which revealed that the 18S rRNA gene sequence of PGSN was 98% similar to that of P. pseudoginseng subsp. himalalaicus var . angustifolius and 96% similar to that of the model plant A. thaliana. Conclusion The use of informations obtained from the model plant, A. thaliana may promote the research progress of molecular biology of TCM drugs.

ARABIDOPSIS GENOME SEQUENCING AND PLANT FUNCTIONAL GENOMICS / 微生物学通报

The first flowering plant genome sequence of Arabidopsis thaliana was completed at the end of 2000.This will greatly promote the studies on systematic identification of functional genes and their expression profiles and the development of technology for functional genomics.