不同产地浙贝母生物碱含量及其合成相关基因表达研究

为了探究浙贝母(Fritillaria thunbergii)药效成分积累量与生物碱合成相关基因表达水平之间的关系,该研究采用UPLC-MS、qPCR技术分别测定不同产地11个样品中总生物碱(贝母素甲和贝母素乙之和)含量和3个参与生物碱合成途径相关基因(HMGR、FPS和DXR)的表达量,同时运用生物统计学方法分析成熟期鳞茎生物碱含量与各基因表达量之间的相关性。结果表明:不同产地浙贝母成熟期鳞茎总生物碱含量存在显著差异(P<0.05),为0.2105% ～ 0.4612%; HMGR和FPS基因在盛花期组织表达、盛花期至成熟期鳞茎表达变化趋势同生物碱含量变化趋势基本一致; DXR基因在成熟期鳞茎中表达量最高,盛花期组织表达、盛花期至成熟期鳞茎表达变化趋势同生物碱含量变化趋势大体不一致; HMGR、FPS基因表达量分别与贝母素甲、贝母素乙和总生物碱含量呈显著或极显著正相关性(P<0.05或P<0.01),以FPS基因表达量与生物碱含量相关系数为最高,相关系数分别为0.672,0.631,0.664,DXR基因与生物碱含量呈低度相关性。由此可以推断,生物碱的积累受MVA途径中HMGR、FPS基因协同调控或者修饰作用明显,受MEP途径中DXR基因调控作用不明显。     

Increased accumulation of the cardio-cerebrovascular disease treatment drug tanshinone in Salvia miltiorrhiza hairy roots by the enzymes 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-d-xylulose 5-phosphate reductoisomerase

Tanshinone is widely used for treatment of cardio-cerebrovascular diseases with increasing demand. Herein, key enzyme genes SmHMGR (3-hydroxy-3-methylglutaryl CoA reductase) and SmDXR (1-deoxy-d-xylulose 5-phosphate reductoisomerase) involved in the tanshinone biosynthetic pathway were introduced into Salvia miltiorrhiza (Sm) hairy roots to enhance tanshinone production. Over-expression of SmHMGR or SmDXR in hairy root lines can significantly enhance the yield of tanshinone. Transgenic hairy root lines co-expressing HMGR and DXR (HD lines) produced evidently higher levels of total tanshinone (TT) compared with the control and single gene transformed lines. The highest tanshinone production was observed in HD42 with the concentration of 3.25 mg g^?1 DW. Furthermore, the transgenic hairy roots showed higher antioxidant activity than control. In addition, transgenic hairy root harboring HMGR and DXR (HD42) exhibited higher tanshinone content after elicitation by yeast extract and/or Ag⁺ than before. Tanshinone can be significantly enhanced to 5.858, 6.716, and 4.426 mg g^?1 DW by YE, Ag⁺, and YE-Ag⁺ treatment compared with non-induced HD42, respectively. The content of cryptotanshinone and dihydrotanshinone was effectively elevated upon elicitor treatments, whereas there was no obvious promotion effect for the other two compounds tanshinone I and tanshinone IIA. Our results provide a useful strategy to improve tanshinone content as well as other natural active products by combination of genetic engineering with elicitors.     

A functional (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase exhibits diurnal regulation of expression in Stevia rebaudiana (Bertoni)

The leaves of stevia [Stevia rebaudiana (Bertoni)] are a rich source of steviol glycosides that are used as non-calorific sweetener in many countries around the world. Steviol moiety of steviol glycosides is synthesized via plastidial 2C-methyl-D-erythritol 4-phosphate pathway, where (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the key enzyme. HDR catalyzes the simultaneous conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into five carbon isoprenoid units, isopentenyl diphosphate and dimethylallyl diphosphate. Stevia HDR (SrHDR) successfully rescued HDR lethal mutant strain MG1655 ara<>ispH upon genetic complementation, suggesting SrHDR to encode a functional protein. The gene exhibited diurnal variation in expression. To identify the possible regulatory elements, upstream region of the gene was cloned and putative cis-acting elements were detected by in silico analysis. Electrophoretic mobility shift assay, using a putative light responsive element GATA showed the binding of nuclear proteins (NP) isolated from leaves during light period of the day, but not with the NP from leaves during the dark period. Data suggested the involvement of GATA box in light mediated gene regulation of SrHDR in stevia.     

Copy number variations of functional genes influence contents of glycyrrhizic acid in Glycyrrhiza uralensis

Glycyrrhiza uralensis is a widely used Chinese herb and glycyrrhizic acid is believed to be its marker compound. Three key enzymes, 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), squalene synthase (SQS) and beta-amyrin synthase (β-AS), are involved in the glycyrrhizic acid biosynthetic pathway. In this paper, the relationship between copy number variations (CNVs) of HMGR, SQS and β-AS genes and the content levels of glycyrrhizic acid in G. uralensis were investigated. CNVs of the 62 G. uralensis samples from different origins were determined by real-time PCR and their glycyrrhizic acid contents were analyzed by HPLC. The real-time PCR results showed that the copy numbers of HMGR, SQS1 and β-AS in the 62 G. uralensis samples were either one copy or two copies. According to the copy number patterns of HMGR, SQS1 and β-AS, the 62 G. uralensis samples can be divided into six groups. Among the six groups, group B with two copies of HMGR, one copy of SQS1 and β-AS contained relatively higher contents of glycyrrhizic acid. The accumulation of glycyrrhizic acid was lower in the group C with two copies of β-AS, one copy of SQS1 and HMGR. The results of this work may provide a basis for enhancing the accumulation of glycyrrhizic acid in cultivars of G. uralensis.     

Over‐expression of DXS gene enhances terpenoidal secondary metabolite accumulation in rose‐scented geranium and Withania somnifera: active involvement of plastid isoprenogenic pathway in their biosynthesis

Rose‐scented geranium (Pelargonium spp.) is one of the most important aromatic plants and is well known for its diverse perfumery uses. Its economic importance is due to presence of fragrance rich essential oil in its foliage. The essential oil is a mixture of various volatile phytochemicals which are mainly terpenes (isoprenoids) in nature. In this study, on the geranium foliage genes related to isoprenoid biosynthesis (DXS, DXR and HMGR) were isolated, cloned and confirmed by sequencing. Further, the first gene of 2‐C‐methyl‐d ‐erythritol‐4‐phosphate (MEP) pathway, 1‐deoxy‐d ‐xylulose‐5‐phosphate synthase (GrDXS), was made full length by using rapid amplification of cDNA ends strategy. GrDXS contained a 2157 bp open reading frame that encoded a polypeptide of 792 amino acids having calculated molecular weight 77.5 kDa. This study is first report on heterologous expression and kinetic characterization of any gene from this economically important plant. Expression analysis of these genes was performed in different tissues as well as at different developmental stages of leaves. In response to external elicitors, such as methyl jasmonate, salicylic acid, light and wounding, all the three genes showed differential expression profiles. Further GrDXS was over expressed in the homologous (rose‐scented geranium) as well as in heterologous (Withania somnifera) plant systems through genetic transformation approach. The over‐expression of GrDXS led to enhanced secondary metabolites production (i.e. essential oil in rose‐scented geranium and withanolides in W. somnifera). To the best of our knowledge, this is the first report showing the expression profile of the three genes related to isoprenoid biosynthesis pathways operated in rose‐scented geranium as well as functional characterization study of any gene from rose‐scented geranium through a genetic transformation system.     

酮型广藿香MVA途径基因表达分析及与主要成分合成相关性研究

广藿香药材以广藿香酮含量较高的酮型广藿香为最优质。而广藿香酮为一种萜类成分,其生物合成途径尚未明确。MVA(甲羟戊酸)途径是萜类化合物生物合成的重要途径。为了分析MVA途经基因表达与化学成分的相关性从而获得促进广藿香酮合成的潜在基因,该文以2种酮型广藿香栽培品种(石牌广藿香、高要广藿香)为材料,通过实时定量PCR分析基因表达和主要成分含量测定,并研究了供试材料不同时期的茎、叶中与甲羟戊酸代谢途径相关的HMGR、MK、MDD基因表达及化学成分。结果表明：(1)HMGR基因在石牌广藿香嫩叶中表达更明显;MK基因在石牌广藿香和高要广藿香中表达模式相似,主要在老茎中表达;MDD基因在石牌广藿香叶中比高要广藿香表达量更高,在两种广藿香的茎中表达模式相似。(2)同属于酮型广藿香,石牌广藿香与高要广藿香的化学成分相似,老叶广藿香醇含量最高,老茎的广藿香酮含量更高。(3)MDD和MK基因与广藿香酮的合成正相关。综上结果所述,酮型广藿香两个栽培种MVA途径的基因表达模式相似,MDD和MK基因可能为酮型广藿香萜类代谢途径的关键基因。     

Isolation and characterization of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene from <Emphasis Type="Italic">Botryococcus braunii</Emphasis>, race B

The B race of a green microalga Botryococcus braunii Kützing produces triterpene hydrocarbons that is a promising source for biofuel. In this algal race, precursors of triterpene hydrocarbons are provided from the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. The terminal enzyme of this pathway, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is regarded as one of the key enzymes that affect yields of products in terpene biosynthesis. In order to better understand the MEP pathway of the alga, cDNA and genomic clones of HDR were obtained from B. braunii Showa strain. B. braunii HDR (BbHDR) is encoded on a single copy gene including a 1509-bp open reading frame that was intervened by 6 introns. The exon–intron structure of BbHDR genes did not show clear relation to phylogeny, while its amino acid sequence reflected phyla and classes well. BbHDR sequence was distinctive from that of the HDR protein from Escherichia coli in the residues involved in hydrogen-bond network that surrounds substrate. Introduction of BbHDR cDNA into an E. coli HDR deficient mutant resulted in recovery of its auxotrophy. BbHDR expression level was upregulated from the onset of liquid culture to the 24th day after inoculation with a 2.5-fold increase and retained its level in the subsequent period.     

Validation and application of reference genes for quantitative gene expression analyses in various tissues of <Emphasis Type="Italic">Bupleurum chinense</Emphasis>

It is crucial to select stable references in gene expression analyses using quantitative real-time PCR (qRT-PCR). In this work, seven frequently used reference genes, 18S, Actin, EF1α, α-tubulin, β-tubulin, Cyclophilin and Cytoplasmic ribosomal protein L2 (L2), from Bupleurum chinense DC. were evaluated as the internal control in five tissues, roots, stems, leaves, flowers and fruits, before tissue specific gene expression assays. The results showed that β-tubulin was the most stable and reliable reference gene among the seven candidate genes in the measured tissues. The expression levels of four genes involved in saikosaponins (the pharmacological active compounds of B. chinense) biosynthesis, HMGR, IPPI, FPS and β-AS, were assayed with β-tubulin as the internal control in the five tissues. All the four genes were expressed in the five tissues with different profiles and HMGR in the order of roots > flowers, stems and leaves > fruits, IPPI of stems > leaves and fruits > roots and flowers, FPS of flowers > fruits > stems and roots > leaves and β-AS of roots > flowers, stems and fruits > leaves. The genes of FPS and β-AS were expressed predominantly in flowers and roots, respectively. This study may provide a suitable internal control for quantitative gene expression assays in various tissues and give insight into the tissue expression profiles of four saikosaponins biosynthesis-involved genes of medicinal B. chinense.     

The SUD1 Gene Encodes a Putative E3 Ubiquitin Ligase and Is a Positive Regulator of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Arabidopsis

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum–associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals.     

Cloning and expression profile of 1-deoxy-D-xylulose 5-phosphate reductoisomerase gene from an oil-bearing rose

The components of rose essential oil are mainly monoterpene alcohols, predominantly synthesized through the methylerythritol 4-phosphate (MEP) pathway in plants. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is specified to be a first committed enzyme of the MEP pathway. In order to understand better the role of DXR in the rose essential oil biosynthesis at the molecular level, the full-length cDNA of DXR sequence (designated as RhDXR) was isolated from an oil-bearing rose hybrid Rosa cv. Zizhi and characterized, and the expression profile of it was investigated. Essential oils of rose cv. Zizhi and the other five oil-bearing roses were distilled to evaluate the relationship between the expression of DXR gene and oil yield rate. The full-length cDNA of RhDXR was 1915 bp in length, comprised an open reading frame of 1419 bp, encoding an enzyme of 472 amino acids. A comparative analysis with DXRs of selected species from bacteria to higher plants revealed three conserved domains: a conserved cleavage site for plastids, an extended Prorich region, and a highly conserved NADPH oxidase-binding motif existing in the N-terminal region, like in other higher plant species. The relative expression levels of the DXR gene were determined in various tissues: receptacle, leaf, sepal, pistil, stamen, and petal (in the order of decreasing expression level), and at different flowering stages (flower bud, flower in half bloom, and flower in full bloom). Six cultivars could be classified into two groups according to flower color, and within each group there was a positive correlation between the expression level of DXR gene and oil yield rate.     

Production of acrylic acid and propionic acid by constructing a portion of the 3-hydroxypropionate/4-hydroxybutyrate cycle from <Emphasis Type="Italic">Metallosphaera sedula</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Acrylic acid and propionic acid are important chemicals requiring affordable, renewable production solutions. Here, we metabolically engineered Escherichia coli with genes encoding components of the 3-hydroxypropionate/4-hydroxybutyrate cycle from Metallosphaera sedula for conversion of glucose to acrylic and propionic acids. To construct an acrylic acid-producing pathway in E. coli, heterologous expression of malonyl-CoA reductase (MCR), malonate semialdehyde reductase (MSR), 3-hydroxypropionyl-CoA synthetase (3HPCS), and 3-hydroxypropionyl-CoA dehydratase (3HPCD) from M. sedula was accompanied by overexpression of succinyl-CoA synthetase (SCS) from E. coli. The engineered strain produced 13.28 ± 0.12 mg/L of acrylic acid. To construct a propionic acid-producing pathway, the same five genes were expressed, with the addition of M. sedula acryloyl-CoA reductase (ACR). The engineered strain produced 1430 ± 30 mg/L of propionic acid. This approach can be expanded to synthesize many important organic chemicals, creating new opportunities for the production of chemicals by carbon dioxide fixation.     

The GATA and SORLIP motifs in the 3-hydroxy-3-methylglutaryl-CoA reductase promoter of Picrorhiza kurrooa for the control of light-mediated expression

Light upregulates the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) in Picrorhiza kurrooa, an endangered medicinal herb. Upstream sequences of HMGR of P. kurrooa (PropkHMGR) were analyzed in relation to its role in light-mediated regulation of gene expression. GATA motif in PropkHMGR exhibited stronger DNA-protein interaction with the nuclear extract of dark-exposed plants in contrast to SORLIP that exhibited stronger binding with the nuclear extract of light-exposed plants. Analysis of PropkHMGR (PropkHMGR-D1, ?1,059/?1) and its deletion fragments PropkHMGR-D2 (?825/?1), PropkHMGR-D3 (?651/?1), PropkHMGR-D4 (?452/?1), and PropkHMGR-D5 (?101/?1) in Arabidopsis thaliana showed PropkHMGR to regulate gene expression [β-glucuronidase (GUS) was used as a reporter gene] at all the developmental stages but only in actively dividing tissues, excluding anthers. Whereas, PropkHMGR-D2 regulated GUS expression in relatively older seedlings but the expression was observed only in shoot apical meristem, root tips, and anthers. PropkHMGR-mediated gene expression was higher in dark as compared to that in the light in Arabidopsis across four temperatures studied. As opposed to the results in P. kurrooa, GATA motifs exhibited DNA-protein interaction with nuclear extract of light-exposed plants of Arabidopsis. SORLIP motifs in Arabidopsis also exhibited DNA-protein interaction with nuclear extract of light-exposed plants as in P. kurrooa. Data showed that (1) PropkHMGR regulated light-mediated gene expression and (2) GATA motif exhibited an inverse relationship between strength of DNA-protein interaction and the gene expression whereas the relationship was species specific for SORLIP.     

Molecular cloning,characterization and function analysis of the gene encoding HMG-CoA reductase from <Emphasis Type="Italic">Euphorbia Pekinensis</Emphasis> Rupr

A new full-length cDNA encoding 3-hydroxy-3-methylglutoryl-Coenzyme A reductase (HMGR; EC1.1.1.34), which catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway, was isolated from young leaves of Euphorbia Pekinensis Rupr. by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of HMGR (designated as EpHMGR, GenBank Accession NO. EF062569) was 2,200 bp containing a 1,752 bp ORF encoding 583 amino acids. Bioinformatic analyzes revealed that the deduced EpHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of EpHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that at most two copies of EpHMGR gene existed in E. Pekinensis genome. Tissue expression analysis revealed that EpHMGR expressed strongly in roots, weakly in stems and leaves. The functional colour complementation assay indicated that EpHMGR could accelerate the biosynthesis of carotenoids in the Escherichia coli transformant, demonstrating that EpHMGR plays an influential role in isoprenoid biosynthesis.     

Salicylic acid activates artemisinin biosynthesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

This paper provides evidence that salicylic acid (SA) can activate artemisinin biosynthesis in Artemisia annua L. Exogenous application of SA to A. annua leaves was followed by a burst of reactive oxygen species (ROS) and the conversion of dihydroartemisinic acid into artemisinin. In the 24 h after application, SA application led to a gradual increase in the expression of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene and a temporary peak in the expression of the amorpha-4,11-diene synthase (ADS) gene. However, the expression of the farnesyl diphosphate synthase (FDS) gene and the cytochrome P450 monooxygenase (CYP71AV1) gene showed little change. At 96 h after SA (1.0 mM) treatment, the concentration of artemisinin, artemisinic acid and dihydroartemisinic acid were 54, 127 and 72% higher than that of the control, respectively. Taken together, these results suggest that SA induces artemisinin biosynthesis in at least two ways: by increasing the conversion of dihydroartemisinic acid into artemisinin caused by the burst of ROS, and by up-regulating the expression of genes involved in artemisinin biosynthesis.     

A comprehensive analysis of fifteen genes of steviol glycosides biosynthesis pathway in Stevia rebaudiana (Bertoni)

Stevia [Stevia rebuaidana (Bertoni); family: Asteraceae] is known to yield diterpenoid steviol glycosides (SGs), which are about 300 times sweeter than sugar. The present work analyzed the expression of various genes of the SGs biosynthesis pathway in different organs of the plant in relation to the SGs content. Of the various genes of the pathway, SrDXS, SrDXR, SrCPPS, SrKS, SrKO and three glucosyltransferases namely SrUGT85C2, SrUGT74G1 and SrUGT76G1 were reported from stevia. Here, we report cloning of seven additional full-length cDNA sequences namely, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR, SrIDI and SrGGDPS followed by expression analysis of all the fifteen genes vis-à-vis SGs content analysis. SGs content was highest in the leaf at 3rd node position (node position with reference to the apical leaf as the first leaf) as compared to the leaves at other node positions. Except for SrDXR and SrKO, gene expression was maximum in leaf at 1st node and minimum in leaf at 5th node. The expression of SrKO was highest in leaf at 3rd node while in case of SrDXR expression showed an increase up to 3rd leaf and decrease thereafter. SGs accumulated maximum in leaf tissue followed by stem and root, and similar was the pattern of expression of all the fifteen genes. The genes responded to the modulators of the terpenopids biosynthesis. Gibberellin (GA₃) treatment up-regulated the expression of SrMCT, SrCMK, SrMDS and SrUGT74G1, whereas methyl jasmonate and kinetin treatment down-regulated the expression of all the fifteen genes of the pathway.     

Developmentally Regulated Sesquiterpene Production Confers Resistance to Colletotrichum gloeosporioides in Ripe Pepper Fruits

Sesquiterpenoid capsidiol, exhibiting antifungal activity against pathogenic fungus, is accumulated in infected ripe pepper fruits. In this study, we found a negative relation between the capsidiol level and lesion size in fruits infected with Colletotrichum gloeosporioides, depending on the stage of ripening. To understand the developmental regulation of capsidiol biosynthesis, fungal-induced gene expressions in the isoprenoid biosynthetic pathways were examined in unripe and ripe pepper fruits. The sterol biosynthetic pathway was almost shut down in healthy ripe fruits, showing very low expression of hydroxymethyl glutaryl CoA reductase (HMGR) and squalene synthase (SS) genes. In contrast, genes in the carotenoid pathway were highly expressed in ripe fruits. In the sesquiterpene pathway, 5-epi-aristolochene synthase (EAS), belonging to a sesquiterpene cyclase (STC) family, was significantly induced in the ripe fruits upon fungal infection. Immunoblot and enzyme activity analyses showed that the STCs were induced both in the infected unripe and ripe fruits, while capsidiol was synthesized discriminatively in the ripe fruits, implying diverse enzymatic specificity of multiple STCs. Thereby, to divert sterol biosynthesis into sesquiterpene production, infected fruits were pretreated with an SS inhibitor, zaragozic acid (ZA), resulting in increased levels of capsidiol by more than 2-fold in the ripe fruits, with concurrent reduction of phytosterols. Taken together, the present results suggest that the enhanced expression and activity of EAS in the ripe fruits play an important role in capsidiol production, contributing to the incompatibility between the anthracnose fungus and the ripe pepper fruits.