RESUMEN
Glutathione S-transferases (GSTs) are members of a protein superfamily with diverse physiological functions, including cellular detoxification and protection against oxidative damage. However, there is limited research on GSTs responding to cadmium (Cd) stress. This study classified 46 GST genes in Dendrobium officinale (D. officinale) into nine groups using model construction and domain annotation. Evolutionary analysis revealed nine subfamilies with diverse physical and chemical properties. Prediction of subcellular localization revealed that half of the GST members were located in the cytoplasm. According to the expression analysis of GST family genes responding to Cd stress, DoGST5 responded significantly to Cd stress. Transient expression of DoGST5-GFP in tobacco leaves revealed that DoGST5 was localized in the cytoplasm. DoGST5 overexpression in Arabidopsis enhanced Cd tolerance by reducing Cd-induced H2O2 and O2- levels. These findings demonstrate that DoGST5 plays a critical role in enhancing Cd tolerance by balancing reactive oxygen species (ROS) levels, offering potential applications for improving plant adaptability to heavy metal stress.
Asunto(s)
Cadmio , Dendrobium , Regulación de la Expresión Génica de las Plantas , Glutatión Transferasa , Proteínas de Plantas , Cadmio/toxicidad , Cadmio/metabolismo , Dendrobium/genética , Dendrobium/enzimología , Dendrobium/efectos de los fármacos , Dendrobium/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Filogenia , Estrés Fisiológico/genética , Estrés Fisiológico/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Familia de Multigenes , Genoma de PlantaRESUMEN
BACKGROUND: Dendrobium officinale Kimura et Migo, a renowned traditional Chinese orchid herb esteemed for its significant horticultural and medicinal value, thrives in adverse habitats and contends with various abiotic or biotic stresses. Acid invertases (AINV) are widely considered enzymes involved in regulating sucrose metabolism and have been revealed to participate in plant responses to environmental stress. Although members of AINV gene family have been identified and characterized in multiple plant genomes, detailed information regarding this gene family and its expression patterns remains unknown in D. officinale, despite their significance in polysaccharide biosynthesis. RESULTS: This study systematically analyzed the D. officinale genome and identified four DoAINV genes, which were classified into two subfamilies based on subcellular prediction and phylogenetic analysis. Comparison of gene structures and conserved motifs in DoAINV genes indicated a high-level conservation during their evolution history. The conserved amino acids and domains of DoAINV proteins were identified as pivotal for their functional roles. Additionally, cis-elements associated with responses to abiotic and biotic stress were found to be the most prevalent motif in all DoAINV genes, indicating their responsiveness to stress. Furthermore, bioinformatics analysis of transcriptome data, validated by quantitative real-time reverse transcription PCR (qRT-PCR), revealed distinct organ-specific expression patterns of DoAINV genes across various tissues and in response to abiotic stress. Examination of soluble sugar content and interaction networks provided insights into stress release and sucrose metabolism. CONCLUSIONS: DoAINV genes are implicated in various activities including growth and development, stress response, and polysaccharide biosynthesis. These findings provide valuable insights into the AINV gene amily of D. officinale and will aid in further elucidating the functions of DoAINV genes.
Asunto(s)
Dendrobium , Regulación de la Expresión Génica de las Plantas , Familia de Multigenes , Filogenia , beta-Fructofuranosidasa , Dendrobium/genética , Dendrobium/enzimología , beta-Fructofuranosidasa/genética , beta-Fructofuranosidasa/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Perfilación de la Expresión Génica , Genoma de Planta , Estrés Fisiológico/genética , Genes de PlantasRESUMEN
To investigate the potential roles of stress-activated protein kinase (SAPK) gene family members in Dendrobium officinale, we employed multiple bioinformatics methods to identify the members of this family. The physicochemical properties, chromosomal localization, phylogenetic relationship, gene structure, and cis-acting elements of each D. officinale SAPK (DoSAPK) member were analyzed. In addition, their expression profiles in different tissues and under the low-temperature or salt stress treatment were determined by real-time fluorescence quantitative PCR. The results showed that D. officinale carried eight DoSAPK family members, which belonged to three groups (groups â , â ¡, and â ¢). These genes were located on seven chromosomes, and there were two pairs of genes with replication. The DoSAPK members within the same group had similar gene structures, conserved motifs, and secondary structures. The cis-acting elements in the promoter regions of DoSAPK genes included abundant hormone and stress response elements. DoSAPK family members presented tissue-specific expression in D. officinale. Furthermore, they were differentially expressed under the low-temperature or salt stress treatment, which suggested that they might be involved in the responses to low-temperature and salt stress. Intriguingly, DoSAPK1 might play a role in the abiotic stress responses. The results laid a foundation for in-depth study of the members and roles of the DoSAPK gene family.
Asunto(s)
Dendrobium , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Familia de Multigenes , Filogenia , Proteínas de Plantas , Dendrobium/genética , Dendrobium/enzimología , Proteínas de Plantas/genética , Estrés Fisiológico/genética , FríoRESUMEN
BACKGROUND: DNA methylation is a conserved and important epigenetic modification involved in the regulation of numerous biological processes, including plant development, secondary metabolism, and response to stresses. However, no information is available regarding the identification of cytosine-5 DNA methyltransferase (C5-MTase) and DNA demethylase (dMTase) genes in the orchid Dendrobium officinale. RESULTS: In this study, we performed a genome-wide analysis of DoC5-MTase and DodMTase gene families in D. officinale. Integrated analysis of conserved motifs, gene structures and phylogenetic analysis showed that eight DoC5-MTases were divided into four subfamilies (DoCMT, DoDNMT, DoDRM, DoMET) while three DodMTases were divided into two subfamilies (DoDML3, DoROS1). Multiple cis-acting elements, especially stress-responsive and hormone-responsive ones, were found in the promoter region of DoC5-MTase and DodMTase genes. Furthermore, we investigated the expression profiles of DoC5-MTase and DodMTase in 10 different tissues, as well as their transcript abundance under abiotic stresses (cold and drought) and at the seedling stage, in protocorm-like bodies, shoots, and plantlets. Interestingly, most DoC5-MTases were downregulated whereas DodMTases were upregulated by cold stress. At the seedling stage, DoC5-MTase expression decreased as growth proceeded, but DodMTase expression increased. CONCLUSIONS: These results provide a basis for elucidating the role of DoC5-MTase and DodMTase in secondary metabolite production and responses to abiotic stresses in D. officinale.
Asunto(s)
Metilación de ADN/genética , ADN-Citosina Metilasas/genética , Dendrobium/enzimología , Dendrobium/genética , Oxidorreductasas/genética , Polisacáridos/genética , Polisacáridos/metabolismo , Arabidopsis/genética , ADN-Citosina Metilasas/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Estudio de Asociación del Genoma Completo , Oryza/genética , Oxidorreductasas/metabolismoRESUMEN
Floral scent is a key ornamental trait that determines the quality and commercial value of orchids. Geraniol, an important volatile monoterpene in orchids that attracts pollinators, is also involved in responses to stresses but the geraniol synthase (GES) responsible for its synthesis in the medicinal orchid Dendrobium officinale has not yet been identified. In this study, three potential geraniol synthases were mined from the D. officinale genome. DoGES1, which was localized in chloroplasts, was characterized as a geraniol synthase. DoGES1 was highly expressed in flowers, especially in petals. DoGES1 transcript levels were high in the budding stage of D. officinale flowers at 11:00 a.m. DoGES1 catalyzed geraniol in vitro, and transient expression of DoGES1 in Nicotiana benthamiana leaves resulted in the accumulation of geraniol in vivo. These findings on DoGES1 advance our understanding of geraniol biosynthesis in orchids, and lay the basis for genetic modification of floral scent in D. officinale or in other ornamental orchids.
Asunto(s)
Proteínas de Cloroplastos , Cloroplastos , Dendrobium , Flores , Odorantes , Monoéster Fosfórico Hidrolasas , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Cloroplastos/enzimología , Cloroplastos/genética , Dendrobium/enzimología , Dendrobium/genética , Flores/enzimología , Flores/genética , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Nicotiana/enzimología , Nicotiana/genéticaRESUMEN
Terpene synthase (TPS) is a critical enzyme responsible for the biosynthesis of terpenes, which possess diverse roles in plant growth and development. Although many terpenes have been reported in orchids, limited information is available regarding the genome-wide identification and characterization of the TPS family in the orchid, Dendrobium officinale. By integrating the D. officinale genome and transcriptional data, 34 TPS genes were found in D. officinale. These were divided into four subfamilies (TPS-a, TPS-b, TPS-c, and TPS-e/f). Distinct tempospatial expression profiles of DoTPS genes were observed in 10 organs of D. officinale. Most DoTPS genes were predominantly expressed in flowers, followed by roots and stems. Expression of the majority of DoTPS genes was enhanced following exposure to cold and osmotic stresses. Recombinant DoTPS10 protein, located in chloroplasts, uniquely converted geranyl diphosphate to linalool in vitro. The DoTPS10 gene, which resulted in linalool formation, was highly expressed during all flower developmental stages. Methyl jasmonate significantly up-regulated DoTPS10 expression and linalool accumulation. These results simultaneously provide valuable insight into understanding the roles of the TPS family and lay a basis for further studies on the regulation of terpenoid biosynthesis by DoTPS in D. officinale.
Asunto(s)
Monoterpenos Acíclicos/metabolismo , Transferasas Alquil y Aril/metabolismo , Dendrobium/enzimología , Proteínas de Plantas/metabolismo , Transferasas Alquil y Aril/genética , Dendrobium/genética , Regulación de la Expresión Génica de las Plantas , Familia de Multigenes , Proteínas de Plantas/genética , Estrés FisiológicoRESUMEN
Four Dendrobium Sonia 'Earsakul' lines were generated by insertion of one, two or three antisense copies of a Carica papaya gene encoding 1-aminocyclopropane-1-carboxylic acid oxidase (CpACO). Whole vegetative plants of the transgenic lines showed about 50% of the basal ethylene production rate, while the increase in ethylene production in floral buds during opening and open flowers prior to visible senescence was delayed. Detailed analysis of more than 100 parameters in flowering plants showed no effect of antisense ACO on plant morphology and coloration, except for shorter length and width of some of the sepals and petals. In intact plants the water-soaking of floral buds as well as bud abscission were delayed by ACO antisense, as was the time to senescence of open flowers. Pollen viability and pollen tube growth were not affected in the transgenic lines. In cut inflorescences placed in water, bud yellowing, bud water soaking, and bud abscission were considerably delayed by the antisense construct, while the life span of open flowers were increased and abscission of open flowers were delayed. It is concluded that the reduction of ACO activity affected the shape of some petals/sepals and delayed the abortion in floral buds, and the senescence and abscission of open flowers.
Asunto(s)
Aminoácido Oxidorreductasas/antagonistas & inhibidores , Elementos sin Sentido (Genética) , Senescencia Celular , Dendrobium/enzimología , Flores/anatomía & histología , Flores/fisiología , Regulación Enzimológica de la Expresión Génica , Aminoácido Oxidorreductasas/genética , ADN de Plantas/genética , Dendrobium/genética , Dendrobium/crecimiento & desarrollo , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrolloRESUMEN
Stigmatic mucilage plays a crucial role in pollen-grain adhesion on the stigma in flowering plants. Little information is available regarding mucilage biosynthesis in orchid plants. In the present study, stigmatic mucilage is rich in galactose-containing polysaccharides, mainly consisted of galactose and arabinose in Dendrobium officinale. Thirteen galactosyltransferases involved in biosynthesis of the ß-1,3-galactose linkage polysaccharides, belonging to the CAZY GT31 family, were identified from D. officinale genome. A positive correlation between the mucilage content and the DoGALT2 expression at different stages was observed. DoGALT2 expressed overall sampled tissues with the highest in D. officinale stigmatic mucilage that contributes to pollen adhesion and elongation. DoGALT2 was targeted to Golgi, and had a GALT domain (PF01762) that was homologous to the characterized GALT2 in Arabidopsis. Compared to wild-type Arabidopsis, DoGALT2 overexpressing plants showed a higher content of galactose and galactose-containing alcohol-insoluble residues, and enhanced tolerance to abiotic stress. DoGALT2 complemented Arabidopsis GALT2 mutant (galt2-1), with an equivalent galactose with wild-type Arabidopsis but significantly higher than galt2-1. These findings provide evidence that DoGALT2 might be involved in regulating the biosynthesis of galactose-containing polysaccharides during D. officinale pollen development.
Asunto(s)
Dendrobium/enzimología , Flores/enzimología , Galactosiltransferasas/metabolismo , Genes de Plantas/fisiología , Mucílago de Planta/biosíntesis , Proteínas de Plantas/metabolismo , Arabidopsis , Clonación Molecular , Dendrobium/genética , Dendrobium/metabolismo , Flores/química , Flores/metabolismo , Galactosa/metabolismo , Galactosiltransferasas/genética , Mucílago de Planta/química , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Polisacáridos/análisis , TranscriptomaRESUMEN
In order to understand the function of GDP-mannose pyrophosphorylase(GMPP) function and its regulation in polysaccharide biosynthesis mechanism in Dendrobium. D. huoshanense was used to clone GMPP gene. GMPP gene expression in D. huoshanense,D. officinale and D. moniliforme was also determined by qPCR. The results showed that the length of D. huoshanense GMPP gene c DNA sequence is 1 867 bp,containing 1 245 bp open reading frame(ORF),encoding 415 amino acids. Phylogenetic tree analysis showed that D. huoshanense,D. officinale and D. moniliforme are closely related with GMPP taken into consideration. Bioinformatics analysis demonstrated that GMPP sequence similarity among the three species reached as high as 99%. qPCR results indicated that GMPP genes was highly expressed in stem of D. huoshanense compared with its leaf,flower and root. According to GMPP gene expression profile in D. huoshanense,D. officinale and D. moniliforme grown in Huoshan area,it was clear that GMPP in D. huoshanense showed the highest expression level. Furthermore,our findings of GMPP gene expression profile will facilitate future researches into its polysaccharide biosynthetic mechanism.
Asunto(s)
Dendrobium/genética , Nucleotidiltransferasas/genética , Proteínas de Plantas/genética , Secuencia de Bases , Clonación Molecular , Dendrobium/enzimología , Filogenia , Polisacáridos/biosíntesisRESUMEN
BACKGROUND: Reactive oxygen species (ROS)-induced oxidative damage is responsible for viability loss in plant tissues following cryopreservation. Antioxidants may improve viability by preventing or repairing the injury. OBJECTIVE: This work aimed at studying the effect of catalase (CAT) and pyruvate dehydrogenase (PDH), which are involved in ROS metabolism and are differentially expressed during pollen cryopreservation, for cryopreservation of Dendrobium nobile Lindl. 'Hamana Lake Dream' protocorm-like bodies (PLBs). MATERIALS AND METHODS: Different concentrations of exogenous CAT or PDH were added at the loading, PVS2 treatment, unloading steps during vitrification-cryopreservation of PLBs. Their survival and regeneration were evaluated and correlated with physiological oxidative indexes. RESULTS: PLB survival increased significantly when CAT and PDH were added separately to the unloading solution at a suitable concentration. CAT at 400 U·ml-1 increased PLB survival and regeneration by 33.5 and 14.6 percent respectively. It had no impact on the production of superoxide anion radical (·O2-) and on superoxide dismutase (SOD) activity, but it reduced the hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents and enhanced ascorbic acid (AsA) and endogenous CAT levels compared to PLBs cryopreserved using the standard vitrification protocol (CK1). PDH at 0.1 U·ml-1 significantly improved PLB survival (by 2.5 percent), but it had no marked effect on regeneration compared to the CK1 group. It induced the same variations in ·O2-, AsA and endogenous CAT levels that were observed following CAT addition. However, PDH did not affect the H2O2 and MDA content but significantly increased SOD activity. CONCLUSION: These results indicate that the addition of 400 U·ml-1 CAT and 0.1 U·ml-1 PDH at the unloading step increased survival of cryopreserved PLBs and that this improvement was associated with scavenging of H2O2 and the repair of oxidative damage. Exogenous CAT also significantly improved PLB regeneration after cryopreservation, while PDH had no obvious effect. The effect of exogenous CAT on PLB survival and regeneration was stronger than that of PDH, which may be due to the increased SOD activity by PDH addition.
Asunto(s)
Catalasa/farmacología , Dendrobium , Estrés Oxidativo/efectos de los fármacos , Complejo Piruvato Deshidrogenasa/farmacología , Antioxidantes/farmacología , Catalasa/metabolismo , Criopreservación/métodos , Dendrobium/efectos de los fármacos , Dendrobium/enzimología , Estrés Oxidativo/fisiología , Complejo Piruvato Deshidrogenasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Regeneración/efectos de los fármacos , VitrificaciónRESUMEN
GDP-mannose pyrophosphorylase (GMP) catalyzed the formation of GDP-mannose, which serves as a donor for the biosynthesis of mannose-containing polysaccharides. In this study, three GMP genes from Dendrobium officinale (i.e., DoGMPs) were cloned and analyzed. The putative 1000 bp upstream regulatory region of these DoGMPs was isolated and cis-elements were identified, which indicates their possible role in responses to abiotic stresses. The DoGMP1 protein was shown to be localized in the cytoplasm. To further study the function of the DoGMP1 gene, 35S:DoGMP1 transgenic A. thaliana plants with an enhanced expression level of DoGMP1 were generated. Transgenic plants were indistinguishable from wild-type (WT) plants in tissue culture or in soil. However, the mannose content of the extracted water-soluble polysaccharides increased 67%, 96% and 92% in transgenic lines #1, #2 and #3, respectively more than WT levels. Germination percentage of seeds from transgenic lines was higher than WT seeds and the growth of seedlings from transgenic lines was better than WT seedlings under salinity stress (150 mM NaCl). Our results provide genetic evidence for the involvement of GMP genes in the biosynthesis of mannose-containing polysaccharides and the mediation of GMP genes in the response to salt stress during seed germination and seedling growth.
Asunto(s)
Dendrobium/enzimología , Manosa/metabolismo , Nucleotidiltransferasas/metabolismo , Presión Osmótica , Polisacáridos/metabolismo , Sales (Química)/metabolismo , Estrés Fisiológico , Arabidopsis , Clonación Molecular , Citoplasma/enzimología , Citoplasma/metabolismo , Dendrobium/metabolismo , Regulación de la Expresión Génica de las Plantas , Germinación , Nucleotidiltransferasas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Secuencias Reguladoras de Ácidos Nucleicos , Cloruro de Sodio/metabolismoRESUMEN
Phosphomannomutase (PMM, EC 5.4.2.8) catalyzes the interconversion of mannose-6-phosphate to mannose-1-phosphate, the precursor for the synthesis of GDP-mannose. In this study, the complementary DNA (cDNA) of the Phosphomannomutase (PMM) gene was initially cloned from Dendrobium officinale by RACE method. Transient transform result showed that the DoPMM protein was localized in the cytoplasm. The DoPMM gene was highly expressed in the stems of D. officinale both in vegetative and reproductive developmental stages. The putative promoter was cloned by TAIL-PCR and used for searched cis-elements. Stress-related cis-elements like ABRE, TCA-element, and MBS were found in the promoter regions. The DoPMM gene was up-regulated after treatment with abscisic acid, salicylic acid, cold, polyethylene glycol, and NaCl. The total ascorbic acid (AsA) and polysaccharide content in all of the 35S::DoPMM Arabidopsis thaliana transgenic lines #1, #2, and #5 showed a 40, 39, and 31% increase in AsA and a 77, 22, and 39% increase in polysaccharides, respectively more than wild-type (WT) levels. All three 35S::DoPMM transgenic lines exhibited a higher germination percentage than WT plants when seeded on half-strength MS medium supplemented with 150 mM NaCl or 300 mM mannitol. These results provide genetic evidence for the involvement of PMM genes in the biosynthesis of AsA and polysaccharides and the mediation of PMM genes in abiotic stress tolerance during seed germination in A. thaliana.
Asunto(s)
Dendrobium/enzimología , Germinación , Fosfotransferasas (Fosfomutasas)/genética , Proteínas de Plantas/genética , Ácido Abscísico/metabolismo , Adaptación Fisiológica , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Citoplasma/enzimología , Dendrobium/crecimiento & desarrollo , Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Especificidad de Órganos , Fosfotransferasas (Fosfomutasas)/metabolismo , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas , Transporte de Proteínas , Estrés FisiológicoRESUMEN
Dendrobium officinale is one of the most well-known traditional Chinese medicines, and polysaccharide is its main active ingredient. Many studies have investigated the synthesis and accumulation mechanisms of polysaccharide, but until recently, little was known about the molecular mechanism of how polysaccharide is synthesized because no related genes have been cloned. In this study, we cloned an alkaline/neutral invertase gene from D. officinale (DoNI) by the rapid amplification of cDNA ends (RACE) method. DoNI was 2231 bp long and contained an open reading frame that predicted a 62.8-kDa polypeptide with 554-amino acid residues. An alkaline/neutral invertase conserved domain was predicted from this deduced amino acid sequence, and DoNI had a similar deduced amino acid sequence to Setaria italica and Oryza brachyantha. We also found that DoNI expression in different tissues was closely related to DoNI activity, and more importantly, polysaccharide level. Our results indicate that DoNI is associated with polysaccharide accumulation in D. officinale.
Asunto(s)
Dendrobium/genética , Genes de Plantas , Proteínas de Plantas/genética , Polisacáridos/metabolismo , beta-Fructofuranosidasa/genética , Secuencia Conservada , Dendrobium/enzimología , Sistemas de Lectura Abierta , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Polisacáridos/genética , Dominios Proteicos , beta-Fructofuranosidasa/química , beta-Fructofuranosidasa/metabolismoRESUMEN
Orchids make up about 10% of all seed plant species, have great economical value, and are of specific scientific interest because of their renowned flowers and ecological adaptations. Here, we report the first draft genome sequence of a lithophytic orchid, Dendrobium catenatum. We predict 28,910 protein-coding genes, and find evidence of a whole genome duplication shared with Phalaenopsis. We observed the expansion of many resistance-related genes, suggesting a powerful immune system responsible for adaptation to a wide range of ecological niches. We also discovered extensive duplication of genes involved in glucomannan synthase activities, likely related to the synthesis of medicinal polysaccharides. Expansion of MADS-box gene clades ANR1, StMADS11, and MIKC(*), involved in the regulation of development and growth, suggests that these expansions are associated with the astonishing diversity of plant architecture in the genus Dendrobium. On the contrary, members of the type I MADS box gene family are missing, which might explain the loss of the endospermous seed. The findings reported here will be important for future studies into polysaccharide synthesis, adaptations to diverse environments and flower architecture of Orchidaceae.
Asunto(s)
Evolución Biológica , Dendrobium/enzimología , Dendrobium/genética , Flores/crecimiento & desarrollo , Genoma de Planta , Glicosiltransferasas/genética , Secuencia de Bases , Vías Biosintéticas , Evolución Molecular , Flores/genética , Genes de Plantas , Glicosiltransferasas/metabolismo , Proteínas de Dominio MADS/genética , Familia de Multigenes , Filogenia , Análisis de Secuencia de ADNRESUMEN
Mitogen-activated protein kinases (MAPKs) are important signaling transduction components well conserved in eukaryotes and play essential roles in various physiological, developmental and hormonal responses in plant. In the present study, a MAPK gene, designated as DoMPK4 (GenBank accession No. JX297597), is identified from a rare endangered medicinal orchid species D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The full length cDNA of DoMPK4 is 1 518 bp in length and encoded a 369 aa protein with a molecular weight of 42.42 kD and an isoelectric point of 5.55. DoMPK4 protein contained a serine/threonine protein kinase active site (158-170), a MAP kinase site (71-174), and eight conserved motifs. DoMPK4 had a transmembrane (214-232) but no signal peptide. Multiple sequence alignment showed that DoMPK4 shared high identities (74.9%-80.6%) with MAPK proteins from various plants. Phylogenetic analysis demonstrated that DoMPK4 belonged to group A of the MAPK evolutionary tree, and is closely related to monocots. Real time quantitative PCR (qPCR) analysis revealed that DoMPK4 is differentially expressed among the five organs including leaf, stem, root, seed, and protocorm-like body (PLB). The transcription level of DoMPK4 is the highest in the PLBs with 17.65 fold, followed by seeds, roots, and stems with 5.84, 2.28, and 1.64 fold, respectively. The progressive enhancement of DoMPK4 transcripts in the developing PLBs compared to that in the germinating seeds, suggests a role of DoMPK4 during the development of embryogenic PLBs formation in D. officinale.
Asunto(s)
Dendrobium/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , ADN Complementario/genética , ADN de Plantas/genética , Dendrobium/enzimología , Regulación de la Expresión Génica de las Plantas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Filogenia , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Tallos de la Planta/metabolismo , Semillas/metabolismo , Alineación de SecuenciaRESUMEN
The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate in mavalonic acid pathway, which is the first committed step for isoprenoid biosynthesis in plants. However, it still remains unclear whether HGMR gene plays a role in the isoprenoid biosynthesis in Dendrobium officinale, an endangered epiphytic orchid species. In the present study, a HMGR encoding gene, designed as DoHMGR1 (GenBank accession JX272632), was identified from D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, for the first time. The full length cDNA of DoHMGR1 was 2 071 bp in length and encoded a 562-aa protein with a molecular weight of 59.73 kD and an isoelectric point (pI) of 6.18. The deduced DoHMGR1 protein, like other HMGR proteins, constituted four conserved domains (63-561, 147-551, 268-383 and 124-541) and two transmembrane motifs (42-64 and 85-107). Multiple sequence alignment and phylogenetic analyses demonstrated that DoHMGR1 had high identity (67%-89%) to a number of HMGR genes from various plants and was closely related to Vanda hybrid cultivar, rice and maize monocots. Real time quantitative PCR (qPCR) analysis revealed that DoHMGR1 was expressed in the three included organs. The transcripts were the most abundant in the roots with 2.13 fold over that in the leaves, followed by that in the stems with 1.98 fold. Molecular characterization of DoHMGR1 will be useful for further functional elucidation of the gene involving in isoprenoid biosynthesis pathway in D. officinale.
Asunto(s)
Dendrobium/enzimología , Hidroximetilglutaril-CoA Reductasas/genética , Plantas Medicinales/enzimología , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Dendrobium/genética , Regulación de la Expresión Génica de las Plantas , Hidroximetilglutaril-CoA Reductasas/metabolismo , Peso Molecular , Filogenia , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Tallos de la Planta/enzimología , Tallos de la Planta/genética , Plantas Medicinales/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Using universal primer Ty1-copia retrotransposon RT,43 Ty1-copia like retrotransposon RT with high heterogeneity, stop codon mutation and frameshift mutation were amplified by PCR from genomic DNA of Zhejiang Lin'an (C15) and Yunnan Guangnan (A39) of Dendrobium officinale. The length of these sequences varied from 260 to 266 bp, and was rich in AT and consistency ranged from 47.1% to 97.7%. Different c/s-acting regulatory elements induced by low temperature, heat, light, all kinds of plant growth regulating substances and the starting transcription signals, corresponding to CAAT box, TATA box conserved sequences and some other regulatory elements. When being translated into amino acids, ten sequences presented stop codon mutation, five sequences presented frameshift mutation, and thirty-seven sequences presented conserved sequence "SLYGKQ" mutation. Six categories were identified through phylogenic analysis after alignment analyses of their amino acid sequences, and with other plants (eg. Triticum aestivum, Eleocharis quinqueflora) having high homology, which indicated that horizontal transmission of retrotransposon occurred among the plants in the past.
Asunto(s)
Dendrobium/enzimología , Dendrobium/genética , ADN Polimerasa Dirigida por ARN/genética , Retroelementos/genética , Secuencia de Aminoácidos , Clonación Molecular , Secuencia Conservada , ADN de Plantas/genética , Datos de Secuencia Molecular , Filogenia , ADN Polimerasa Dirigida por ARN/química , TATA Box/genéticaRESUMEN
S-Adenosyl-L-methionine decarboxylase (SAMDC) is a key enzyme in the polyamines biosynthesis, thus is essential for basic physiological and biochemical processes in plant. In the present study, a full length cDNA of DoSAMDC1 gene was obtained from symbiotic germinated seeds of an endangered medicinal orchid species Dendrobium officinale, using the rapid amplification of cDNA ends (RACE)-PCR technique for the first time. The full length cDNA was 1 979 bp, with three open reading frames, i.e. tiny-uORF, small-uORF and main ORF (mORF). The mORF was deduced to encode a 368 amino acid (aa) protein with a molecular mass of 40.7 kD and a theoretical isoelectric point of 5.2. The deduced DoSAMDC1 protein, without signal peptide, had two highly conserved function domains (proenzyme cleavage site and PEST domain) and a 22-aa transmembrane domain (89-110). Multiple sequence alignments and phylogenetic relationship analyses revealed DoSAMDC1 had a higher level of sequence similarity to monocot SAMDCs than those of dicot. Expression patterns using qRT-PCR analyses showed that DoSAMDC1 transcripts were expressed constitutively without significant change in the five tissues (not infected with fungi). While in the symbiotic germinated seeds, the expression level was enhanced by 2.74 fold over that in the none-germinated seeds, indicating possible involvement of the gene in symbiotic seed germination of D. officinale.
Asunto(s)
Adenosilmetionina Descarboxilasa/genética , Dendrobium/genética , Sistemas de Lectura Abierta , Simbiosis , Adenosilmetionina Descarboxilasa/aislamiento & purificación , Secuencia de Aminoácidos , Basidiomycota/fisiología , Clonación Molecular , ADN Complementario/genética , Dendrobium/enzimología , Dendrobium/microbiología , Germinación , Filogenia , Plantas Medicinales/enzimología , Plantas Medicinales/genética , Plantas Medicinales/microbiología , Semillas/genética , Semillas/crecimiento & desarrollo , Semillas/microbiología , Alineación de Secuencia , Simbiosis/fisiologíaRESUMEN
Dendrobium officinale Kimura et Migo (Orchidaceae) is a traditional Chinese medicinal plant. The stem contains an alkaloid that is the primary bioactive component. However, the details of alkaloid biosynthesis have not been effectively explored because of the limited number of expressed sequence tags (ESTs) available in GenBank. In this study, we analyzed RNA isolated from the stem of D. officinale using a single half-run on the Roche 454 GS FLX Titanium platform to generate 553,084 ESTs with an average length of 417 bases. The ESTs were assembled into 36,407 unique putative transcripts. A total of 69.97% of the unique sequences were annotated, and a detailed view of alkaloid biosynthesis was obtained. Functional assignment based on Kyoto Encyclopedia of Genes and Genomes (KEGG) terms revealed 69 unique sequences representing 25 genes involved in alkaloid backbone biosynthesis. A series of qRT-PCR experiments confirmed that the expression levels of 5 key enzyme-encoding genes involved in alkaloid biosynthesis are greater in the leaves of D. officinale than in the stems. Cytochrome P450s, aminotransferases, methyltransferases, multidrug resistance protein (MDR) transporters and transcription factors were screened for possible involvement in alkaloid biosynthesis. Furthermore, a total of 1061 simple sequence repeat motifs (SSR) were detected from 36,407 unigenes. Dinucleotide repeats were the most abundant repeat type. Of these, 179 genes were associated with a metabolic pathway in KEGG. This study is the first to produce a large volume of transcriptome data from D. officinale. It extends the foundation to facilitate gene discovery in D. officinale and provides an important resource for the molecular genetic and functional genomic studies in this species.
Asunto(s)
Dendrobium/genética , Proteínas de Plantas/genética , Transcriptoma , Alcaloides/biosíntesis , Vías Biosintéticas/genética , Dendrobium/enzimología , Marcadores Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Medicina Tradicional China , Anotación de Secuencia Molecular , Proteínas de Plantas/metabolismo , Tallos de la Planta , Plantas Medicinales/enzimología , Plantas Medicinales/genética , Análisis de Secuencia de ADN , Repeticiones de TrinucleótidosRESUMEN
In this study, a phenylalanine ammonia-lyase (PAL) gene was cloned from Dendrobium candidum using homology cloning and RACE. The full-length sequence and catalytic active sites that appear in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum are also found: PAL cDNA of D. candidum (designated Dc-PAL1, GenBank No. JQ765748) has 2,458 bps and contains a complete open reading frame (ORF) of 2,142 bps, which encodes 713 amino acid residues. The amino acid sequence of DcPAL1 has more than 80% sequence identity with the PAL genes of other plants, as indicated by multiple alignments. The dominant sites and catalytic active sites, which are similar to that showing in PAL proteins of Arabidopsis thaliana and Nicotiana tabacum, are also found in DcPAL1. Phylogenetic tree analysis revealed that DcPAL is more closely related to PALs from orchidaceae plants than to those of other plants. The differential expression patterns of PAL in protocorm-like body, leaf, stem, and root, suggest that the PAL gene performs multiple physiological functions in Dendrobium candidum.