Overexpression of AtPYL5 under the control of guard cell specific promoter improves drought stress tolerance in Arabidopsis.

PYR/PYLs function as ABA receptors and are key regulators during plant drought stress response. Previously we screened drought tolerance of Arabidopsis ABA receptors PYR/PYLs under the control of five different promoters. In this study, we characterized drought stress tolerance of AtPYL5 transgene under the control of one guard cell specific promoter, pGC1. pGC1::AtPYL5 transgenic Arabidopsis exhibited reduced transpiration rate and decreased water loss after drought treatment. Transformation of pGC1::AtPYL5 in Arabidopsis also decreased oxidative stress damage and improved photosynthesis under drought stress condition. These results indicated that pGC1::AtPYL5 construct is effective and might pave new way to develop genetically engineered plants to improve drought stress tolerance.

Selection and validation of reference genes for normalization of quantitative real-time reverse transcription PCR analysis in Poria cocos (Schw.) Wolf (Fuling).

BACKGROUND: Quantitative real-time reverse transcription PCR (qRT-PCR) requires a stable internal control to avoid misinterpretation of data or errors for gene expression normalization. However, there are still no validated reference genes for stable internal control in Poria cocos (Schw.) Wolf (Fuling). This study aims to validate the reference genes of P. cocos. METHODS: This study firstly collected the 14 candidate reference genes by BLASTP from the genome of P. cocos for qRT-PCR analysis to determine the expression levels of 14 housekeeping genes (GAPDH, MAPK, ß-Act, RPB2, RPB1-1, RPB1-2, his3-1, his3-2, APT, SAMDC, RP, ß-Tub, EIF, and CYP) under different temperatures and in response to different plant hormones (indole-3-acetic acid, abscisic acid, 6-benzylaminopurine, methyl jasmonate, and gibberellic acid), and the threshold cycle (Ct) values. The results were analyzed by four programs (i.e., geNorm, NormFinder, BestKeeper, and RefFinder) for evaluating the candidate reference genes. RESULTS: SAMDC, his3-2, RP, RPB2, and his3-1 were recommended as reference genes for treating P. cocos with indole-3-acetic acid, abscisic acid, 6-benzylaminopurine, methyl jasmonate, and gibberellic acid, respectively. Under different temperatures RPB2 was the most stable reference gene. CYP was the most stable gene for all 90 samples by RefFinder. CONCLUSION: SAMDC, his3-2, RP, RPB2, and his3-1 were evaluated to be suitable reference genes for P. cocos following different treatments. RPB2 was the most stable reference gene under different temperatures and CYP was the most stable gene in the mycelia under all six evaluated conditions.

ABA receptor PYL9 promotes drought resistance and leaf senescence.

Drought stress is an important environmental factor limiting plant productivity. In this study, we screened drought-resistant transgenic plants from 65 promoter-pyrabactin resistance 1-like (PYL) abscisic acid (ABA) receptor gene combinations and discovered that pRD29A::PYL9 transgenic lines showed dramatically increased drought resistance and drought-induced leaf senescence in both Arabidopsis and rice. Previous studies suggested that ABA promotes senescence by causing ethylene production. However, we found that ABA promotes leaf senescence in an ethylene-independent manner by activating sucrose nonfermenting 1-related protein kinase 2s (SnRK2s), which subsequently phosphorylate ABA-responsive element-binding factors (ABFs) and Related to ABA-Insensitive 3/VP1 (RAV1) transcription factors. The phosphorylated ABFs and RAV1 up-regulate the expression of senescence-associated genes, partly by up-regulating the expression of Oresara 1. The pyl9 and ABA-insensitive 1-1 single mutants, pyl8-1pyl9 double mutant, and snrk2.2/3/6 triple mutant showed reduced ABA-induced leaf senescence relative to the WT, whereas pRD29A::PYL9 transgenic plants showed enhanced ABA-induced leaf senescence. We found that leaf senescence may benefit drought resistance by helping to generate an osmotic potential gradient, which is increased in pRD29A::PYL9 transgenic plants and causes water to preferentially flow to developing tissues. Our results uncover the molecular mechanism of ABA-induced leaf senescence and suggest an important role of PYL9 and leaf senescence in promoting resistance to extreme drought stress.

RDM4 modulates cold stress resistance in Arabidopsis partially through the CBF-mediated pathway.

The C-REPEAT-BINDING FACTOR (CBF) pathway has important roles in plant responses to cold stress. How the CBF genes themselves are activated after cold acclimation remains poorly understood. In this study, we characterized cold tolerance of null mutant of RNA-DIRECTED DNA METHYLATION 4 (RDM4), which encodes a protein that associates with RNA polymerases Pol V and Pol II, and is required for RNA-directed DNA methylation (RdDM) in Arabidopsis. The results showed that dysfunction of RDM4 reduced cold tolerance, as evidenced by decreased survival and increased electrolyte leakage. Mutation of RDM4 resulted in extensive transcriptomic reprogramming. CBFs and CBF regulon genes were down-regulated in rdm4 but not nrpe1 (the largest subunit of PolV) mutants, suggesting that the role of RDM4 in cold stress responses is independent of the RdDM pathway. Overexpression of RDM4 constitutively increased the expression of CBFs and regulon genes and decreased cold-induced membrane injury. A great proportion of genes affected by rdm4 overlapped with those affected by CBFs. Chromatin immunoprecipitation results suggested that RDM4 is important for Pol II occupancy at the promoters of CBF2 and CBF3. We present evidence of a considerable role for RDM4 in regulating gene expression at low temperature, including the CBF pathway in Arabidopsis.

Genome-wide selection of superior reference genes for expression studies in Ganoderma lucidum.

Quantitative real-time polymerase chain reaction (qRT-PCR) is widely used for the accurate analysis of gene expression. However, high homology among gene families might result in unsuitability of reference genes, which leads to the inaccuracy of qRT-PCR analysis. The release of the Ganoderma lucidum genome has triggered numerous studies to be done on the homology among gene families with the purpose of selecting reliable reference genes. Based on the G. lucdum genome and transcriptome database, 38 candidate reference genes including 28 novel genes were systematically selected and evaluated for qRT-PCR normalization. The result indicated that commonly used polyubiquitin (PUB), beta-actin (BAT), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were unsuitable reference genes because of the high sequence similarity and low primer specificity. According to the evaluation of RefFinder, cyclophilin 5 (CYP5) was ranked as the most stable reference gene for 27 tested samples under all experimental conditions and eighteen mycelial samples. Based on sequence analysis and expression analysis, our study suggested that gene characteristic, primer specificity of high homologous genes, allele-specificity expression of candidate genes and under-evaluation of reference genes influenced the accuracy and sensitivity of qRT-PCR analysis. This investigation not only revealed potential factors influencing the unsuitability of reference genes but also selected the superior reference genes from more candidate genes and testing samples than those used in the previous study. Furthermore, our study established a model for reference gene analysis by using the genomic sequence.

[Research progress of the regulation on active compound biosynthesis by the bHLH transcription factors in plants].

Transcription factor is one of the key factors in the regulation of gene expression at the transcriptional level. It plays an important role in plant growth, active components biosynthesis and response to environmental change. This paper summarized the structure and classification of bHLH transcription factors and elaborated the research progress of bHLH transcription factors which regulate the active components in plants, such as flavonoids, alkaloids, and terpenoids. In addition, the possibility of increasing the concentration of active substances by bHLH in medicinal plants was assessed. The paper emphasized great significance of model plants and multidisciplinary research fields including modern genomics, transcriptomics, metabolomics and bioinformatics, providing the contribution to improve the discovery and function characterization of bHLH transcription factors. Accelerating the research in the mechanism of bHLH transcription factors on the regulation of active components biosynthesis will promote the development of breeding and variety improvement of Chinese medicinal materials, also ease the pressure of resources exhaustion of traditional Chinese medicine home and abroad.

Identification and evaluation of reference genes for qRT-PCR normalization in Ganoderma lucidum.

Quantitative real-time reverse transcription PCR (qRT-PCR) is a rapid, sensitive, and reliable technique for gene expression studies. The accuracy and reliability of qRT-PCR results depend on the stability of the reference genes used for gene normalization. Therefore, a systematic process of reference gene evaluation is needed. Ganoderma lucidum is a famous medicinal mushroom in East Asia. In the current study, 10 potential reference genes were selected from the G. lucidum genomic data. The sequences of these genes were manually curated, and primers were designed following strict criteria. The experiment was conducted using qRT-PCR, and the stability of each candidate gene was assessed using four commonly used statistical programs-geNorm, NormFinder, BestKeeper, and RefFinder. According to our results, PP2A was expressed at the most stable levels under different fermentation conditions, and RPL4 was the most stably expressed gene in different tissues. RPL4, PP2A, and ß-tubulin are the most commonly recommended reference genes for normalizing gene expression in the entire sample set. The current study provides a foundation for the further use of qRT-PCR in G. lucidum gene analysis.

[Progress in the study of Velvet and LaeA proteins and their relation to the development and bioactive compounds in medicinal fungi].

The medicinal fungi, which are of great importance in traditional medicine, are facing the problems of wild resources scarcity and low concentration of bioactive compounds. Velvet family and LaeA global regulator play a vital role in secondary metabolism and developmental programs, which are found in a wide variety of fungi ranging from Chytridiomycota to Basidiomycota. This review elaborates the structures and functions between Velvet family and LaeA protein. The Velvet family which shares the Velvet protein domain, including VeA (Velvet), VelB (Velvet like B), VosA (viability of spores A) and VelC (Velvet like C), acts on the regulation function is secondary metabolism and developmental programs such as asexual and sexual development. Furthermore, the function is affected by environmental factors such as light and temperature. LaeA protein which owns S-adenosylmethionine-dependent methyltransferase domain, coordinately regulates development and secondary metabolism by regulating and modifying the Velvet proteins. The regulation of LaeA is mediated by light receptor proteins. Therefore, clarifying the mechanism of Velvet and LaeA proteins in medicinal fungi will pave the way for nurturing medicinal fungi and improving production of bioactive compounds.

Transcriptomic and physiological variations of three Arabidopsis ecotypes in response to salt stress.

Salt stress is one of the major abiotic stresses in agriculture worldwide. Analysis of natural genetic variation in Arabidopsis is an effective approach to characterize candidate salt responsive genes. Differences in salt tolerance of three Arabidopsis ecotypes were compared in this study based on their responses to salt treatments at two developmental stages: seed germination and later growth. The Sha ecotype had higher germination rates, longer roots and less accumulation of superoxide radical and hydrogen peroxide than the Ler and Col ecotypes after short term salt treatment. With long term salt treatment, Sha exhibited higher survival rates and lower electrolyte leakage. Transcriptome analysis revealed that many genes involved in cell wall, photosynthesis, and redox were mainly down-regulated by salinity effects, while transposable element genes, microRNA and biotic stress related genes were significantly changed in comparisons of Sha vs. Ler and Sha vs. Col. Several pathways involved in tricarboxylic acid cycle, hormone metabolism and development, and the Gene Ontology terms involved in response to stress and defense response were enriched after salt treatment, and between Sha and other two ecotypes. Collectively, these results suggest that the Sha ecotype is preconditioned to withstand abiotic stress. Further studies about detailed gene function are needed. These comparative transcriptomic and analytical results also provide insight into the complexity of salt stress tolerance mechanisms.

Oral immunogenicity of potato-derived antigens to Mycobacterium tuberculosis in mice.

The novel use of transgenic plants as vectors for the expression of viral and bacterial antigens has been increasingly tested as an alternative methodology for the production and delivery of experimental oral vaccines. Here, we examined the immunogenicity of combined plant-made vaccines that include four genes encoding immune-dominant antigens from Mycobacterium tuberculosis. Compared with the wild type and other control groups, mice treated with the combined plant-made vaccines showed significantly higher levels of interferon-γ and interleukin-2 production in response to all four proteins, and higher levels of antigen-specific CD4(+) and CD8(+) T-cell responses and immunoglobulin (Ig) G and IgA titers. These results suggest that combined plant-made vaccines can induce immunogenicity against M. tuberculosis through the induction of stronger Th1-associated immune responses. This is the first report of an orally delivered combined plant-made vaccine against tuberculosis priming an antigen-specific Th1 response, a comprehensive effect including both mucosal and systemic immune responses.

[Preliminary study on promoting effects of endophytic fungi to growth of Rehmannia glutinosa].

In previous studies, four endophytic fungi were isolated from different swollen roots of Rehmannia glutinosa. It's thought that Ceratobasidium sp. , one of the discovered endophytic fingi, was a major promoter for the growth of the roots. In this study, symbiotic experiments were performed to measure the effects of different endophytic fingi cultivated with R. glutinosa. The results showed that the R. glutinosa had significant increases in the size of roots and amount of chlorophyll cultivated with Ceratobasidium sp. And it was tested that indoleacetic acid secreted by Ceratobasidium sp. maybe the effective factor for the promotion of the growth.

The modified castor bean catalase intron is incompletely spliced in tobacco and Arabidopsis.

In an attempt to insert the modified castor bean catalase intron (mCBC intron) into the coding sequence of the Cre recombinase gene, we found that the mCBC intron was not completely spliced from the resulting iCre gene in tobacco and Arabidopsis. Sequencing and allele-specific PCR analyses indicated that six nucleotides (UUACAG) at the 3' terminus of the mCBC intron were retained in the mature mRNA of the iCre gene. Moreover, the mCBC intron was incompletely spliced from the Gus gene in pCAMBIA vectors. A mutational analysis of the mCBC intron demonstrated that the incomplete splicing was due to an artificial 3' splice site introduced by the insertion of an adenine, which created a TAG (stop) codon near the 3' splice site of the original CBC intron. Deletion of the inserted adenine or the six nucleotides that were retained from the mCBC intron led to the complete removal of the intron from the resulting iCre2 and iCre3 genes. Thus, in this study, we not only characterized the incomplete splicing event of the mCBC intron in tobacco and Arabidopsis, but also reported the construction of two intron-containing Cre recombinase genes that are useful for plant biotechnology applications.

A phytocyanin-related early nodulin-like gene, BcBCP1, cloned from Boea crassifolia enhances osmotic tolerance in transgenic tobacco.

Using the mRNA differential display combined with 5' rapid amplification of cDNA ends (RACE) technique, an early nodulin-like protein gene (BcBCP1) (accession no. AY243047.1) was isolated from drought-treated Boea crassifolia leaves. The full-length cDNA of BcBCP1 consists of 844 bp nucleotides and has an open reading frame of 606 bp, encoding a putative polypeptide of 201 amino acids with a predicted molecular mass of 22 kDa and a pI of 5.13. The putative protein precursor contains four sequence domains, including a 27 amino acid hydrophobic N-terminal transit peptide, a 100 amino acid phytocyanin-homologous globular domain, a 51 amino acid hydroxyproline-rich cell wall structural protein domain, and a 22 amino acid hydrophobic extension domain. Sequence alignment defined the encoded protein as an early nodulin-like protein, and the absence of key ligands implies that it is unlikely to bind copper. BcBCP1 expression was strongly induced by dehydration, salinity and abscisic acid (ABA), slightly induced by moderate heat shock, and weakly inhibited by low temperature, methyl jasmonic acid (MeJA), and a low concentration of salicylic acid (SA). Overexpression of BcBCP1 in tobacco under the control of CaMV 35S promoter enhanced tolerance to osmotic stress, as indicated by the less impaired growth, less damaged membrane integrity and lower lipid peroxidation levels after osmotic stress. Transgenic tobacco lines overexpressing BcBCP1 showed higher photosynthetic rates, higher antioxidant enzyme activities and higher cytosyl ascorbic peroxidase transcription levels than non-transgenic tobacco plants, both under normal conditions and under osmotic stress.

Vitreoscilla hemoglobin (VHb) overexpression increases hypoxia tolerance in zebrafish (Danio rerio).

Aquaculture farming may benefit from genetically engineering fish to tolerate environmental stress. Here, we used the vector pCVCG expressing the Vitreoscilla hemoglobin (vhb) gene driven by the common carp ß-actin promoter to create stable transgenic zebrafish. The survival rate of the 7-day-old F(2) transgenic fish was significantly greater than that of the sibling controls under 2.5% O(2) (dissolved oxygen (DO), 0.91 mg/l). Meanwhile, we investigated the relative expression levels of several marker genes (hypoxia-inducible factor alpha 1, heat shock cognate 70-kDa protein, erythropoietin, beta and alpha globin genes, lactate dehydrogenase, catalase, superoxide dismutase, and glutathione peroxidase) of transgenic fish and siblings after hypoxia exposure for 156 h. The expression profiles of the vhb transgenic zebrafish revealed that VHb could partially alleviate the hypoxia stress response to improve the survival rate of the fish. These results suggest that that vhb gene may be an efficient candidate for genetically modifying hypoxia tolerance in fish.

An SGS3-like protein functions in RNA-directed DNA methylation and transcriptional gene silencing in Arabidopsis.

RNA-directed DNA methylation (RdDM) is an important epigenetic mechanism for silencing transgenes and endogenous repetitive sequences such as transposons. The RD29A promoter-driven LUCIFERASE transgene and its corresponding endogenous RD29A gene are hypermethylated and silenced in the Arabidopsis DNA demethylase mutant ros1. By screening for second-site suppressors of ros1, we identified the RDM12 locus. The rdm12 mutation releases the silencing of the RD29A-LUC transgene and the endogenous RD29A gene by reducing the promoter DNA methylation. The rdm12 mutation also reduces DNA methylation at endogenous RdDM target loci, including transposons and other repetitive sequences. In addition, the rdm12 mutation affects the levels of small interfering RNAs (siRNAs) from some of the RdDM target loci. RDM12 encodes a protein with XS and coiled-coil domains, and is similar to SGS3, which is a partner protein of RDR6 and can bind to double-stranded RNAs with a 5' overhang, and is required for several post-transcriptional gene silencing pathways. Our results show that RDM12 is a component of the RdDM pathway, and suggest that RdDM may involve double-stranded RNAs with a 5' overhang and the partnering between RDM12 and RDR2.

Isolation and characterization of a gene encoding cinnamate 4-hydroxylase from Parthenocissus henryana.

Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) plays an important role in the phenylpropanoid pathway, which produces many economically important secondary metabolites. A gene coding for C4H, designated as PhC4H (GenBank accession no. DQ211885) was isolated from Parthenocissus henryana. The full-length PhC4H cDNA is 1,747 bp long with a 1,518-bp open reading frame encoding a protein of 505 amino acids, a 40-bp 5' non-coding region and a 189-bp 3'-untranslated region. Secondary structure of the deduced PhC4H protein consists of 41.78% alpha helix, 15.64% extended strand and 42.57% random coil. The genomic DNA of PhC4H is 2,895 bp long and contains two introns; intron I is 205-bp and intron II is 1,172-bp (GenBank accession no. EU440734). DNA gel blot analysis revealed that there might be a single copy of PhC4H in Parthenocissus henryana genome. By using anchored PCR, a 963-bp promoter sequence was isolated and it contains many responsive elements conserved in the upstream region of PAL, C4H and 4CL including the P-, A-, L- and H-boxes.

Molecular cloning and characterization of a gene encoding RING zinc finger ankyrin protein from drought-tolerant Artemisia desertorum.

A RING zinc finger ankyrin protein gene,designated AdZFP1, was isolated from drought-tolerant Artemisia desertorum Spreng by mRNA differential display and RACE. Its cDNA was 1723 bp and encoded a putative protein of 445 amino acids with a predicted molecular mass of 47.9 kDa and an isoelectric point (pI) of 7.49. A typical C3HC4- type RING finger domain was found at the C-terminal region of the AdZFP1 protein,and several groups of ankyrin repeats were found at the N-terminal region. Alignments of amino acid sequence showed that AdZFP1 was 66% identical to the Arabidopsis thaliana putative RING zinc finger ankyrin protein AAN31869. Transcriptional analysis showed that AdZFP1 was inducible under drought stress in root,stem and leaf of the plant.Semi-quantitative reverse- transcriptase-polymerase chain reaction (RT-PCR) analysis showed that the transcript of AdZFP1 was strongly induced by exogenous abscisic acid (ABA) and also by salinity,cold and heat to some extent. Overexpression of the AdZFP1 gene in transgenic tobacco enhanced their tolerance to drought stress.

Computational analysis of the relationship between allergenicity and digestibility of allergenic proteins in simulated gastric fluid.

BACKGROUND: Safety assessment of genetically modified (GM) food, with regard to allergenic potential of transgene-encoded xenoproteins, typically involves several different methods, evaluation by digestibility being one thereof. However, there are still debates about whether the allergenicity of food allergens is related to their resistance to digestion by the gastric fluid. The disagreements may in part stem from classification of allergens only by their sources, which we believe is inadequate, and the difficulties in achieving identical experimental conditions for studying digestion by simulated gastric fluid (SGF) so that results can be compared. Here, we reclassify allergenic food allergens into alimentary canal-sensitized (ACS) and non-alimentary canal-sensitized (NACS) allergens and use a computational model that simulates gastric fluid digestion to analyze the digestibilities of these two types. RESULTS: The model presented in this paper is as effective as SGF digestion experiments, but more stable and reproducible. On the basis of this model, food allergens are satisfactorily classified as ACS and NACS types by their pathways for sensitization; the former are relatively resistant to gastric fluid digestion while the later are relatively labile. CONCLUSION: The results suggest that it is better to classify allergens into ACS and NACS types when understanding the relationship between their digestibility and allergenicity and the digestibility of a target foreign protein is a parameter for evaluating its allergenicity during safety assessments of GM food.

Expression and characteristic of synthetic human epidermal growth factor (hEGF) in transgenic tobacco plants.

To improve the accumulation of recombinant human epidermal growth factor (hEGF) in transgenic tobacco, a highly effective vector was constructed and transformed via Agrobacterium tumefaciens. The hEGF content in transgenic tobacco was up to 0.3% of the total soluble protein. Using the Vero E6 cell expansion assay and the MTT method for cell proliferation, hEGF produced by transgenic tobacco significantly stimulated Vero E6 cell expansion and proliferation, the same as commercial hEGF products.

[Advance of plant symbiosis receptor-like kinase in nonlegumes].

Most plants can form a symbiosis in root with microorganisms for mutual benefit, Nonlegumes mainly form the symbiotic mycorrhiza with arbuscular fungi. The interaction is initiated by invasion of arbuscular mycorrhizal (AM) fungi into the plant root, and follows by production of several special signal molecules, such as the symbiosis receptor-like kinase (SYMRK) from plant. SYMRK has an extracellular domain comprising three leucine-rich repeats (LRRs), a transmembrane domain and an cytoplasmic protein kinase domain. Symrk is required for a symbiotic signal transduction pathway from the perception of microbial signal molecules to the rapid symbiosis-related gene activation. Study of symrk may set up a solid foundation for giving further insight on the function and mechanism of plant-fungi symbiosis.