Dear Editor,Artemisinin,which has potent antimalarial properties,is a sesquiterpene endoperoxide originally isolated from the traditional Chinese medicinal plant Artemisia annua.However,the artemisinin content in wild...Dear Editor,Artemisinin,which has potent antimalarial properties,is a sesquiterpene endoperoxide originally isolated from the traditional Chinese medicinal plant Artemisia annua.However,the artemisinin content in wild-type(WT)A.annua is low(1-10 mg/g dry weight),leading to its erratic supply and price fluctuations[1].展开更多
We investigated the effects of graphene on the model herb Artemisia annua,which is renowned for produc-ing artemisinin,a widely used pharmacological compound.Seedling growth and biomass were promoted when A.annua was ...We investigated the effects of graphene on the model herb Artemisia annua,which is renowned for produc-ing artemisinin,a widely used pharmacological compound.Seedling growth and biomass were promoted when A.annua was cultivated with low concentrations of graphene,an effect which was attributed to a 1.4-fold increase in nitrogen uptake,a 15%–22%increase in chlorophyllfluorescence,and greater abun-dance of carbon cycling–related bacteria.Exposure to 10 or 20 mg/L graphene resulted in a�60%increase in H2O2,and graphene could act as a catalyst accelerator,leading to a 9-fold increase in catalase(CAT)ac-tivity in vitro and thereby maintaining reactive oxygen species(ROS)homeostasis.Importantly,graphene exposure led to an 80%increase in the density of glandular secreting trichomes(GSTs),in which artemisinin is biosynthesized and stored.This contributed to a 5%increase in artemisinin content inmature leaves.Inter-estingly,expression of miR828 was reduced by both graphene and H2O2 treatments,resulting in induction of its target gene AaMYB17,a positive regulator of GST initiation.Subsequent molecular and genetic assays showed that graphene-induced H2O2 inhibits micro-RNA(miRNA)biogenesis through Dicers and regulates the miR828–AaMYB17 module,thus affecting GST density.Our results suggest that graphene may contribute to yield improvement in A.annua via dynamic physiological processes together with miRNA regulation,and it may thus represent a new cultivation strategy for increasing yield capacity through nanobiotechnology.展开更多
The ＇double T-DNA＇ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene （hpf） in one T-DNA region and three target genes （hLF, SB401, RZ10） in another T-DNA r...The ＇double T-DNA＇ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene （hpf） in one T-DNA region and three target genes （hLF, SB401, RZ10） in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for anther culture. RT-PCR analysis indicated that target genes were inserted in rice genomic DNA and successfully transcribed. It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes with co-transformation followed by anther culture technique, and the efficiency was 12.2%. It was also noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.展开更多
Catharanthus roseus contains important anti-tumor terpenoid indole alkaloids (TIAs) such as vinblastine and vincristine. Cytochrome P450 enzyme geraniol 10-hydroxylase (G10H) is a putative rate-limiting enzyme involve...Catharanthus roseus contains important anti-tumor terpenoid indole alkaloids (TIAs) such as vinblastine and vincristine. Cytochrome P450 enzyme geraniol 10-hydroxylase (G10H) is a putative rate-limiting enzyme involved in the TIAs biosynthetic pathway in C. roseus. In this study the g10h gene driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter was introduced into C. roseus through Agrobacterium-mediated transformation. The integration and overexpression of the target gene (g10h) in hairy root lines were confirmed by polymerase chain reaction and RT-QPCR analysis respectively. Overexpression of g10h in transgenic hairy root lines significantly enhanced the accumulations of monomeric alkaloid ajmalicine and dimeric alkaloids, vincristine and vinblastine. Total TIAs production in hairy roots reached (9.51) mg/g DW, over 3-fold higher than that in the untransformed root lines. This is the first report that engineering of g10h into TIAs-producing plant species results in significant enhancement of TIAs accumulation in cultured hairy roots. This study demonstrates that the putative rate-limiting step catalyzed by G10H is indeed the real rate-limiting step involved in the TIAs biosynthetic pathway in C. roseus, which is one of the key targets for promoting TIAs production by genetic engineering.展开更多
Artemisia annua is a medicinal plant rich in terpenes and flavonoids with useful biological activities such as antioxidant,anticancer,and antimalarial activities.The transcriptional regulation of flavonoid biosynthesi...Artemisia annua is a medicinal plant rich in terpenes and flavonoids with useful biological activities such as antioxidant,anticancer,and antimalarial activities.The transcriptional regulation of flavonoid biosynthesis in A.annua has not been well-studied.In this study,we identified a YABBY family transcription factor,AaYABBY5,as a positive regulator of anthocyanin and total flavonoid contents in A.annua.AaYABBY5 was selected based on its similar expression pattern to the phenylalanine ammonia lyase(PAL),chalcone synthase(CHS),chalcone isomerase(CHI),and flavonol synthase(FLS)genes.A transient dual-luciferase assay in Nicotiana bethamiana with the AaYABBY5 effector showed a significant increase in the activity of the downstream LUC gene,with reporters AaPAL,AaCHS,AaCHI,and AaUFGT.The yeast one-hybrid system further confirmed the direct activation of these promoters by AaYABBY5.Gene expression analysis of stably transformed AaYABBY5 overexpression,AaYABBY5 antisense,and control plants revealed a significant increase in the expression of AaPAL,AaCHS,AaCHI,AaFLS,AaFSII,AaLDOX,and AaUFGT in AaYABBY5 overexpression plants.Moreover,their total flavonoid content and anthocyanin content were also found to increase.AaYABBY5 antisense plants showed a significant decrease in the expression of flavonoid biosynthetic genes,as well as a decrease in anthocyanin and total flavonoid contents.In addition,phenotypic analysis revealed deep purple-pigmented stems,an increase in the leaf lamina size,and higher trichome densities in AaYABBY5 overexpression plants.Together,these data proved that AaYABBY5 is a positive regulator of flavonoid biosynthesis in A.annua.Our study provides candidate transcription factors for the improvement of flavonoid concentrations in A.annua and can be further extended to elucidate its mechanism of regulating trichome development.展开更多
Artemisia annua,a traditional Chinese medicinal plant,remains the only plant source for artemisinin production,yet few genes have been identified to be involved in both the response to biotic stresses,such as pathogen...Artemisia annua,a traditional Chinese medicinal plant,remains the only plant source for artemisinin production,yet few genes have been identified to be involved in both the response to biotic stresses,such as pathogens,and artemisinin biosynthesis.Here,we isolated and identified the WRKY transcription factor(TF)AaWRKY17,which could significantly increase the artemisinin content and resistance to Pseudomonas syringae in A.annua.Yeast one-hybrid(Y1H),dual-luciferase(dual-LUC),and electrophoretic mobility shift assay(EMSA)results showed that AaWRKY17 directly bound to the W-box motifs in the promoter region of the artemisinin biosynthetic pathway gene amorpha-4,11-diene synthase(ADS)and promoted its expression.Real-time quantitative PCR(RT-qPCR)analysis revealed that the transcript levels of two defense marker genes,Pathogenesis-Related 5(PR5)and NDR1/HIN1-LIKE 10(NHL10),were greatly increased in AaWRKY17-overexpressing transgenic A.annua plants.Additionally,overexpression of AaWRKY17 in A.annua resulted in decreased susceptibility to P.syringae.These results indicated that AaWRKY17 acted as a positive regulator in response to P.syringae infection.Together,our findings demonstrated that the novel WRKY transcription factor AaWRKY17 could potentially be used in transgenic breeding to improve the content of artemisinin and pathogen tolerance in A.annua.展开更多
The sesquiterpene lactone artemisinin is an important anti-malarial component produced by the glandular secretory trichomes of sweet wormwood(Artemisia annua L.).Light was previously shown to promote artemisinin produ...The sesquiterpene lactone artemisinin is an important anti-malarial component produced by the glandular secretory trichomes of sweet wormwood(Artemisia annua L.).Light was previously shown to promote artemisinin production,but the underlying regulatory mechanism remains elusive.In this study,we demonstrate that ELONGATED HYPOCOTYL5(HY5),a central transcription factor in the light signaling pathway,cannot promote artemisinin biosynthesis on its own,as the binding of AaHY5 to the promoters of artemisinin biosynthetic genes failed to activate their transcription.Transcriptome analysis and yeast two-hybrid screening revealed the B-box transcription factor AaBBX21 as a potential interactor with AaHY5.AaBBX21 showed a trichome-specific expression pattern.Additionally,the AaBBX21–AaHY5 complex cooperatively activated transcription from the promoters of the downstream genes AaGSW1,AaMYB108,and AaORA,encoding positive regulators of artemisinin biosynthesis.Moreover,AaHY5 and AaBBX21 physically interacted with the A.annua E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1(COP1).In the dark,AaCOP1 decreased the accumulation of AaHY5 and AaBBX21 and repressed the activation of genes downstream of the AaHY5–AaBBX21 complex,explaining the enhanced production of artemisinin upon light exposure.Our study provides insights into the central regulatory mechanism by which light governs terpenoid biosynthesis in the plant kingdom.展开更多
Metal ions are essential for plant growth and development,but in excess,these compounds can become highly toxic.Plants have adopted numerous ways to maintain metal homeostasis while mitigating adverse effects of exces...Metal ions are essential for plant growth and development,but in excess,these compounds can become highly toxic.Plants have adopted numerous ways to maintain metal homeostasis while mitigating adverse effects of excess metal ions,including phytochelatin and the metal-chelating proteins metallothioneins(MTs).A family of cysteine(Cys)-rich,intracellular,and low-molecular-weight(4-8 kDa)MTs are proteins found in nearly all phyla including plants,animals,and fungi,and they have the potential to scavenge reactive oxygen species and detoxify toxic metals including copper,cadmium,and zinc.Based on their Cys numbers and residues,MTs have been categorized into three major classes.Class I MTs,which have highly conserved Cys residues,are found in animals,while class II MTs,with less conserved Cys residues,are present in plants and are classified further into four groups.Class III MTs include phytochelatins,a group of enzymatically synthesized Cys-rich proteins.The MTs have been an area of interest for five decades with extensive studies,which have been facilitated by advancements in instrumental techniques,protein science,and molecular biology tools.Here,we reviewed current advances in our understanding of the regulation of MT biosynthesis,their expression,and their potential roles in the alleviation of abiotic stresses(i.e.,drought,salinity,and oxidative stresses)and heavy metal detoxification and homeostasis.展开更多
As a member of an important group of lipid soluble antioxidants, tocopherols play a paramount role in the daily diet of humans and animals. Recently, genes required for tocochromanol biosynthesis pathway have been ide...As a member of an important group of lipid soluble antioxidants, tocopherols play a paramount role in the daily diet of humans and animals. Recently, genes required for tocochromanol biosynthesis pathway have been identified and cloned with the help of genomics-based approaches and molecular manipulation in the model organisms： Arabidopsis thaliana and Synechocystis sp. PCC 6803. At the basis of these foundations, genetic manipulation of tocochromanol biosynthesis pathway can give rise to strategies that enhance the level of tocochromanol content or convert the constitution of tocochromanol. In addition, genetic manipulations of the tocochromanol biosynthesis pathway provide help for the study of the function of tocopherol in plant systems. The present article summarizes recent advances and pays special attention to the functions of tocopherol in plants. The roles of tocopherol in the network of reactiv.e oxygen species, antioxidants and phytohormones to maintain redox homeostasis and the functions of tocopherol as a signal molecule in chloroplast-to- nucleus signaling to regulate carbohydrate metabolism are also discussed.展开更多
Soils and ecosystems contaminated with cadmium (Cd) threaten human health and adversely affect morphological,physiological,and biochemical parameters of plants.The symbiotic association of endophytic fungi with their ...Soils and ecosystems contaminated with cadmium (Cd) threaten human health and adversely affect morphological,physiological,and biochemical parameters of plants.The symbiotic association of endophytic fungi with their host plants is the best strategy to improve various plant characteristics and remediate soils polluted with heavy metal(loid)s (HMs).Being a well-known plant growth-promoting fungus,Piriformospora indica confers resistance against a number of abiotic stresses,including HM stress.This pot experiment explored the potential and ameliorative effects of P.indica on Artemisia annua L.plants treated with different concentrations (0,40,80,and 120 mg kg-1) of Cd.Inoculation with P.indica significantly increased plant performance,especially by enhancing chlorophyll concentration and water potential and by decreasing electrolytic leakage,when compared with un-inoculated plants,despite the high Cd levels.Similarly,P.indica enhanced antioxidant enzyme activities,thereby reducing the drastic effects of Cd in inoculated plants.In addition,P.indica accumulated Cd in the roots of colonized plants,as revealed by atomic absorption spectroscopy,and restricted Cd translocation to aerial parts.Furthermore,P.indica showed in vitro resistance (up to a certain level) to Cd stress;however,fungus growth was inhibited at very high Cd concentrations,proving it an excellent candidate for use as a potential phytoremediator in fields affected by Cd contamination.The transcriptional analysis showed that the signaling genes and artemisinin and flavonoid biosynthetic pathway genes were significantly upregulated in P.indica-co-cultivated plants when compared with un-inoculated plants,suggesting a fine collaboration between primary and secondary metabolisms to modulate resistance capacity and to enhance the phytoremediation capability of A.annua against Cd toxicity.展开更多
Trace metals such as manganese(Mn),copper(Cu),zinc(Zn),and iron(Fe)are essential for many biological processes in plant life cycles.However,in excess,they can be toxic and disrupt plant growth processes,which is econo...Trace metals such as manganese(Mn),copper(Cu),zinc(Zn),and iron(Fe)are essential for many biological processes in plant life cycles.However,in excess,they can be toxic and disrupt plant growth processes,which is economically undesirable for crop production.For this reason,processes such as homeostasis and transport control of these trace metals are of constant interest to scientists studying heavily contaminated habitats.Phytoremediation is a promising cleanup technology for soils polluted with heavy metals.However,this technique has some disadvantages,such as the slow growth rate of metal-accumulating plant species,low bioavailability of heavy metals,and long duration of remediation.Microbial-assisted phytoremediation is a promising strategy for hyperaccumulating,detoxifying,or remediating soil contaminants.Arbuscular mycorrhizal fungi(AMF)are found in association with almost all plants,contributing to their healthy performance and providing resistance against environmental stresses.They colonize plant roots and extend their hyphae to the rhizosphere region,assisting in mineral nutrient uptake and regulation of heavy metal acquisition.Endophytic fungi exist in every healthy plant tissue and provide enormous services to their host plants,including growth enhancement by nutrient acquisition,detoxification of heavy metals,secondary metabolite regulation,and enhancement of abiotic/biotic stress tolerance.The aim of the present work is to review the recent literature regarding the role of AMF and endophytic fungi in plant heavy metal tolerance in terms of its regulation in highly contaminated conditions.展开更多
Artemisia annua, commonly known as sweet wormwood or Qinghao, is a shrub native to China and has long been used for medicinal purposes. A. annua is now cultivated globally as the only natural source of a potent anti-m...Artemisia annua, commonly known as sweet wormwood or Qinghao, is a shrub native to China and has long been used for medicinal purposes. A. annua is now cultivated globally as the only natural source of a potent anti-malarial compound, artemisinin. Here, we report a high-quality draft assembly of the 1.74-gigabase genome of A. annua, which is highly heterozygous, rich in repetitive sequences, and contains 63 226 protein-coding genes, one of the largest numbers among the sequenced plant species. We found that, as one of a few sequenced genomes in the Asteraceae, the A. annua genome contains a large number of genes specific to this large angiosperm clade. Notably, the expansion and functional diversification of genes encoding enzymes involved in terpene biosynthesis are consistent with the evolution of the artemi- sinin biosynthetic pathway. We further revealed by transcriptome profiling that A. annua has evolved the sophisticated transcriptional regulatory networks underlying artemisinin biosynthesis. Based on compre- hensive genomic and transcriptomic analyses we generated transgenic A. annua lines producing high levels of artemisinin, which are now ready for large-scale production and thereby will help meet the chal- lenge of increasing global demand of artemisinin.展开更多
Artemisinin, also known as qinghaosu, a sesquiterpene endoperoxide lactone isolated from the Chinese medicinal plant Artemisia annua L., is the most effective antimalarial drug which has saved millions of lives.Due to...Artemisinin, also known as qinghaosu, a sesquiterpene endoperoxide lactone isolated from the Chinese medicinal plant Artemisia annua L., is the most effective antimalarial drug which has saved millions of lives.Due to its great antimalarial activity and low content in wild A. annua plants, researches focused on enhancing the artemisin yield in plants became a hotspot. Several families of transcription factors have been reported to participate in regulating the biosynthesis and accumulation of artemisinin.In this review, we summarize recent investigations in these fields, with emphasis on newly identified transcription factors and their functions in artemisinin biosynthesis regulation, and provide new insight for further research.展开更多
The voltage-dependent anion channel （VDAC） plays an essential role in the permeability of mltochondrial membrane. In the present study, we isolated a novel VDAC gene （brvdac） based on the assembly of expressed seq...The voltage-dependent anion channel （VDAC） plays an essential role in the permeability of mltochondrial membrane. In the present study, we isolated a novel VDAC gene （brvdac） based on the assembly of expressed sequence tag sequences from Brassica rapa L. and explored its differential expression patterns In growth, tissues, abiotlc stress, and stress recovery. Results of a tissue-specific expression study in young seedlings Indicated that, of all tissues tested, brvdac expression was the highest in the leaves. Under cold, drought, and salt stresses, brvdac expression showed a transient Increase, and then returned to normal levels when the stress was removed. When plants were exposed to heat shock, there was no Increase in brvdac expression, whereas during recovery a quick and considerable increase in expression was observed. These observations indicate that dissimilar modulations of brvdactranscription may occur when plant cells encounter heat shock and the other three types of stress. In addition, phylogenetic analysis Implied that an earlier duplication of vdac probably occurred before the divergence between monocotyledons and dicotyledons.展开更多
The important antimalarial drug artemisinin is biosynthesized and stored in Artemisia annua glandular trichomes and the artemisinin content correlates with trichome density;however,the factors affecting trichome devel...The important antimalarial drug artemisinin is biosynthesized and stored in Artemisia annua glandular trichomes and the artemisinin content correlates with trichome density;however,the factors affecting trichome development are largely unknown.Here,we demonstrate that the A.annua R2R3 MYB transcription factor TrichomeLess Regulator 1(TLR1)negatively regulates trichome development.In A.annua,TLR1 overexpression lines had 44.7%–64.0%lower trichome density and 11.5%–49.4%lower artemisinin contents and TLR1-RNAi lines had 33%–93.3%higher trichome density and 32.2%–84.0%higher artemisinin contents compared with non-transgenic controls.TLR1 also negatively regulates the expression of anthocyanin biosynthetic pathway genes in A.annua.When heterologously expressed in Arabidopsis thaliana,TLR1 interacts with GLABROUS3a,positive regulator of trichome development,and represses trichome development.Yeast two-hybrid and pull-down assays indicated that TLR1 interacts with the WUSCHEL homeobox(WOX)protein AaWOX1,which interacts with the LEAFY-like transcription factor TLR2.TLR2 overexpression in Arabidopsis and A.annua showed that TLR2 reduces trichome development by reducing gibberellin levels.Furthermore,artemisinin contents were 19%–43%lower in TLR2-overexpressing A.annua plants compared to controls.These data indicate that TLR1 and TLR2 negatively regulate trichome density by lowering gibberellin levels and may enable approaches to enhance artemisinin yields.展开更多
A homeodomain leucine-zipper （HD-Zip） gene BnHB6 （GenBank accession No. AY336103） was isolated from oilseed rape （Brassica napus L.） following drought treatment through rapid amplification of cDNA ends （RACE）....A homeodomain leucine-zipper （HD-Zip） gene BnHB6 （GenBank accession No. AY336103） was isolated from oilseed rape （Brassica napus L.） following drought treatment through rapid amplification of cDNA ends （RACE）. The full-length cDNA of BnHB6 was 1 611 bp and contained a 936-bp open reading frame encoding 311 amino acids. Sequence analysis indicated that BnHB6 belonged to the HD-Zip I subfamily. High-stringency Southern boltting analysis showed that BnHB6 appeared in rape as a single copy but had homologous genes. Semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） analysis revealed that BnHB6 was expressed in several tissues tested under control conditions, but that expression was significantly upregulated in shoots by mannitol, NaCI, cold treatment, anaerobic culture, wounding, H2O2, abscisic acid （ABA）, and salicylic acid （SA） treatments, but not by ultraviolet treatment. Further RT- PCR analysis revealed that BnHB6 was a late-responsive gene, the expression of which was not activated by NaCI, cold treatment, H2O2, ABA, and SA at an early time point （20 min） of treatment in the shoot. However, after a certain period of treatment, the induced expression culminated and then declined until the next peak occurred. Tissue-specific analysis revealed that BnHB6 was expressed at certain levels in the roots, shoots, and flowers, and the roots were found to respond to the osmotic stimuli more rapidly than shoots to increase the expression of BnHB6. The present study implies that BnHB6 plays a positive role as a regulator of biotic and abiotic stresses on growth during seedling establishment.展开更多
Catharanthus roseus L. （Madagascar periwinkle） biosynthesizes a diverse array of secondary metabolites including anticancer dimeric alkaloids （vinblastine and vincristine） and antihypertensive alkaloids （ajmalici...Catharanthus roseus L. （Madagascar periwinkle） biosynthesizes a diverse array of secondary metabolites including anticancer dimeric alkaloids （vinblastine and vincristine） and antihypertensive alkaloids （ajmalicine and serpentine）. The multi-step terpenoid indole alkaloids （TIAs） biosynthetic pathway in C. roseus is complex and is under strict molecular regulation. Many enzymes and genes involved in the TIAs biosynthesis have been studied in recent decades. Moreover, some regulatory proteins were found recently to control the production of TIAs in C. roseus. Based on mastering the rough scheme of the pathway and cloning the related genes, metabolic engineering of TIAs biosynthesis has been studied in C. roseus aiming at increasing the desired secondary metabolites in the past few years. The present article summarizes recent advances in isolation and characterization of TIAs biosynthesis genes and transcriptional regulators involved in the second metabolic control in C. roseus. Metabolic engineering applications in TIAs pathway via overexpression of these genes and regulators in C. roseus are also discussed.展开更多
Farnesyl dlphosphate synthase （FPS; EC 2.5.1.10） catalyzes the production of 15-carbon farnesyl dlphosphate which Is a branch-point Intermediate for many terpenoids. This reaction Is considered to be a ratelimiting ...Farnesyl dlphosphate synthase （FPS; EC 2.5.1.10） catalyzes the production of 15-carbon farnesyl dlphosphate which Is a branch-point Intermediate for many terpenoids. This reaction Is considered to be a ratelimiting step In terpenold biosynthesis. Here we report for the first time the cloning of a new full-length cDNA encoding farnesyl dlphosphate synthase from a gymnosperm plant species, Taxus media Rehder, designated as TmFPS1. The full-length cDNA of TmFPS1 （GenBank accession number： AY461811） was 1 464 bp with a 1 056-bp open reading frame encoding a 351-amino acid polypeptlde with a calculated molecular weight of 40.3 kDa and a theoretical pl of 5.07. Biolnformatlc analysis revealed that TmFPS1 contained all five conserved domains of prenyltransferases, and showed homology to other FPSs of plant origin. Phylogenetlc analysis showed that farnesyl dlphosphate synthases can be divided Into two groups： one of prokaryotic origin and the other of eukaryotic origin. TmFPS1 was grouped with FPSs of plant origin. Homologybased structural modeling showed that TmFPS1 had the typical spatial structure of FPS, whose most prominent structural feature Is the arrangement of 13 core helices around a large central cavity In which the catalytic reaction takes place. Our blolnformatic analysis strongly suggests that TmFPS1 is a functional gene. Southern blot analysis revealed that TmFPS1 belongs to a small FPSgene family in T. media. Northern blot analysis indicated that TmFPS1 is expressed in all tested tissues, Including the needles, stems and roots of T. media. Subsequently, functional complementatlon with TmFPS1 in a FPS-deflclent mutant yeast demonstrated that TmFPS1 did encode farnesyl dlphosphate synthase, which rescued the yeast mutant. This study will be helpful In future Investigations aiming at understanding the detailed role of FPS In terpenold biosynthesis flux control at the molecular genetic level.展开更多
Metabolomics has been rapidly developed as an important field in plant sciences and natural products chemistry.As the only natural source for a diversity of monoterpenoid indole alkaloids(MIAs),especially the low-abun...Metabolomics has been rapidly developed as an important field in plant sciences and natural products chemistry.As the only natural source for a diversity of monoterpenoid indole alkaloids(MIAs),especially the low-abundance antitumor agents vinblastine and vincristine,Catharanthus roseus is highly valued and has been studied extensively as a model for medicinal plants improvement.Due to multistep enzymatic biosynthesis and complex regulation,genetic modification in the MIA pathway has resulted in complicated changes of both secondary and primary metabolism in C.roseus,affecting not only the MIA pathway but also other pathways.Research at the metabolic level is necessary to increase knowledge on the genetic regulation of the whole metabolic network connected to MIA biosynthesis.Nuclear magnetic resonance(NMR)is a very suitable and powerful complementary technique for the identification and quantification of metabolites in the plant matrix.NMR-based metabolomics has been used in studies of C.roseus for pathway elucidation,understanding stress responses,classification among different cultivars,safety and quality controls of transgenic plants,cross talk between pathways,and diversion of carbon fluxes,with the aim of fully unravelling MIA biosynthesis,its regulation and the function of the alkaloids in the plant from a systems biology point of view.展开更多
基金This work was supported by National Key R&D Program of China(2018YFA0900600)the Bill&Melinda Gates Foundation(OPP1199872 and INV-027291)+6 种基金the China Postdoctoral Science Foundation(2022M722851)the National Natural Science Foundation of China(82274047,31770327,32070329,82003889)SJTU Trans-med Awards Research(20190104)SJTU Global Strategic Partnership Fund(2020 SJTU-CORNELL)Zhejiang Provincial Natural Science Foundation of China(LQ21H280004)National Young Qihuang Scholars Training Programthe National‘Ten-thousand Talents Program’for Leading Talents of Science and Technology Innovation in China.
文摘Dear Editor,Artemisinin,which has potent antimalarial properties,is a sesquiterpene endoperoxide originally isolated from the traditional Chinese medicinal plant Artemisia annua.However,the artemisinin content in wild-type(WT)A.annua is low(1-10 mg/g dry weight),leading to its erratic supply and price fluctuations[1].
基金supported by the National Natural Science Foundation of China (82274047 and 52071192)the National Key R&D Program of China (2018YFA0900600)+3 种基金the Engineering Research Center of Coal-Based Ecological Carbon Sequestration Technology of the Ministry of Education (MJST2023-3)the China Postdoctoral Science Foundation (2023M732232)SJTU Trans-med Awards Research (20190104)supported in part by the Bill&Melinda Gates Foundation (OPP1199872 and INV-027291).
文摘We investigated the effects of graphene on the model herb Artemisia annua,which is renowned for produc-ing artemisinin,a widely used pharmacological compound.Seedling growth and biomass were promoted when A.annua was cultivated with low concentrations of graphene,an effect which was attributed to a 1.4-fold increase in nitrogen uptake,a 15%–22%increase in chlorophyllfluorescence,and greater abun-dance of carbon cycling–related bacteria.Exposure to 10 or 20 mg/L graphene resulted in a�60%increase in H2O2,and graphene could act as a catalyst accelerator,leading to a 9-fold increase in catalase(CAT)ac-tivity in vitro and thereby maintaining reactive oxygen species(ROS)homeostasis.Importantly,graphene exposure led to an 80%increase in the density of glandular secreting trichomes(GSTs),in which artemisinin is biosynthesized and stored.This contributed to a 5%increase in artemisinin content inmature leaves.Inter-estingly,expression of miR828 was reduced by both graphene and H2O2 treatments,resulting in induction of its target gene AaMYB17,a positive regulator of GST initiation.Subsequent molecular and genetic assays showed that graphene-induced H2O2 inhibits micro-RNA(miRNA)biogenesis through Dicers and regulates the miR828–AaMYB17 module,thus affecting GST density.Our results suggest that graphene may contribute to yield improvement in A.annua via dynamic physiological processes together with miRNA regulation,and it may thus represent a new cultivation strategy for increasing yield capacity through nanobiotechnology.
文摘The ＇double T-DNA＇ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene （hpf） in one T-DNA region and three target genes （hLF, SB401, RZ10） in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for anther culture. RT-PCR analysis indicated that target genes were inserted in rice genomic DNA and successfully transcribed. It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes with co-transformation followed by anther culture technique, and the efficiency was 12.2%. It was also noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.
基金Item supported by China national"863"high-tech program (2002AA212191)China ministry of educa-tion and science and technology commission of Shanghai(04XD14011)
文摘Catharanthus roseus contains important anti-tumor terpenoid indole alkaloids (TIAs) such as vinblastine and vincristine. Cytochrome P450 enzyme geraniol 10-hydroxylase (G10H) is a putative rate-limiting enzyme involved in the TIAs biosynthetic pathway in C. roseus. In this study the g10h gene driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter was introduced into C. roseus through Agrobacterium-mediated transformation. The integration and overexpression of the target gene (g10h) in hairy root lines were confirmed by polymerase chain reaction and RT-QPCR analysis respectively. Overexpression of g10h in transgenic hairy root lines significantly enhanced the accumulations of monomeric alkaloid ajmalicine and dimeric alkaloids, vincristine and vinblastine. Total TIAs production in hairy roots reached (9.51) mg/g DW, over 3-fold higher than that in the untransformed root lines. This is the first report that engineering of g10h into TIAs-producing plant species results in significant enhancement of TIAs accumulation in cultured hairy roots. This study demonstrates that the putative rate-limiting step catalyzed by G10H is indeed the real rate-limiting step involved in the TIAs biosynthetic pathway in C. roseus, which is one of the key targets for promoting TIAs production by genetic engineering.
基金This work was supported by the National Key R&D Program of China(2018YFA0900600)the Bill&Melinda Gates Foundation(OPP1199872)+1 种基金the SJTU Global Strategic Partnership Fund(2020SJTU-CORNELL)the SJTU Trans-med Awards Research Program(20190104).
文摘Artemisia annua is a medicinal plant rich in terpenes and flavonoids with useful biological activities such as antioxidant,anticancer,and antimalarial activities.The transcriptional regulation of flavonoid biosynthesis in A.annua has not been well-studied.In this study,we identified a YABBY family transcription factor,AaYABBY5,as a positive regulator of anthocyanin and total flavonoid contents in A.annua.AaYABBY5 was selected based on its similar expression pattern to the phenylalanine ammonia lyase(PAL),chalcone synthase(CHS),chalcone isomerase(CHI),and flavonol synthase(FLS)genes.A transient dual-luciferase assay in Nicotiana bethamiana with the AaYABBY5 effector showed a significant increase in the activity of the downstream LUC gene,with reporters AaPAL,AaCHS,AaCHI,and AaUFGT.The yeast one-hybrid system further confirmed the direct activation of these promoters by AaYABBY5.Gene expression analysis of stably transformed AaYABBY5 overexpression,AaYABBY5 antisense,and control plants revealed a significant increase in the expression of AaPAL,AaCHS,AaCHI,AaFLS,AaFSII,AaLDOX,and AaUFGT in AaYABBY5 overexpression plants.Moreover,their total flavonoid content and anthocyanin content were also found to increase.AaYABBY5 antisense plants showed a significant decrease in the expression of flavonoid biosynthetic genes,as well as a decrease in anthocyanin and total flavonoid contents.In addition,phenotypic analysis revealed deep purple-pigmented stems,an increase in the leaf lamina size,and higher trichome densities in AaYABBY5 overexpression plants.Together,these data proved that AaYABBY5 is a positive regulator of flavonoid biosynthesis in A.annua.Our study provides candidate transcription factors for the improvement of flavonoid concentrations in A.annua and can be further extended to elucidate its mechanism of regulating trichome development.
基金This work was supported by the National Key R&D Program of China(2018YFA0900600)the Bill&Melinda Gates Foundation(OPP1199872 and INV-027291)+1 种基金SJTU Trans-med Awards Research(20190104)the SJTU Global Strategic Partnership Fund(2020 SJTU-CORNELL).
文摘Artemisia annua,a traditional Chinese medicinal plant,remains the only plant source for artemisinin production,yet few genes have been identified to be involved in both the response to biotic stresses,such as pathogens,and artemisinin biosynthesis.Here,we isolated and identified the WRKY transcription factor(TF)AaWRKY17,which could significantly increase the artemisinin content and resistance to Pseudomonas syringae in A.annua.Yeast one-hybrid(Y1H),dual-luciferase(dual-LUC),and electrophoretic mobility shift assay(EMSA)results showed that AaWRKY17 directly bound to the W-box motifs in the promoter region of the artemisinin biosynthetic pathway gene amorpha-4,11-diene synthase(ADS)and promoted its expression.Real-time quantitative PCR(RT-qPCR)analysis revealed that the transcript levels of two defense marker genes,Pathogenesis-Related 5(PR5)and NDR1/HIN1-LIKE 10(NHL10),were greatly increased in AaWRKY17-overexpressing transgenic A.annua plants.Additionally,overexpression of AaWRKY17 in A.annua resulted in decreased susceptibility to P.syringae.These results indicated that AaWRKY17 acted as a positive regulator in response to P.syringae infection.Together,our findings demonstrated that the novel WRKY transcription factor AaWRKY17 could potentially be used in transgenic breeding to improve the content of artemisinin and pathogen tolerance in A.annua.
基金supported by the National Natural Science Foundation of China (32070329, 31770327)Shanghai Natural Science Foundation (16ZR1418000)the National Key R&D Program of China (2018Y FA0900600)。
文摘The sesquiterpene lactone artemisinin is an important anti-malarial component produced by the glandular secretory trichomes of sweet wormwood(Artemisia annua L.).Light was previously shown to promote artemisinin production,but the underlying regulatory mechanism remains elusive.In this study,we demonstrate that ELONGATED HYPOCOTYL5(HY5),a central transcription factor in the light signaling pathway,cannot promote artemisinin biosynthesis on its own,as the binding of AaHY5 to the promoters of artemisinin biosynthetic genes failed to activate their transcription.Transcriptome analysis and yeast two-hybrid screening revealed the B-box transcription factor AaBBX21 as a potential interactor with AaHY5.AaBBX21 showed a trichome-specific expression pattern.Additionally,the AaBBX21–AaHY5 complex cooperatively activated transcription from the promoters of the downstream genes AaGSW1,AaMYB108,and AaORA,encoding positive regulators of artemisinin biosynthesis.Moreover,AaHY5 and AaBBX21 physically interacted with the A.annua E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1(COP1).In the dark,AaCOP1 decreased the accumulation of AaHY5 and AaBBX21 and repressed the activation of genes downstream of the AaHY5–AaBBX21 complex,explaining the enhanced production of artemisinin upon light exposure.Our study provides insights into the central regulatory mechanism by which light governs terpenoid biosynthesis in the plant kingdom.
基金This research was supported by the National Key R&D Programe of China(No.2018 YFA 0900600).
文摘Metal ions are essential for plant growth and development,but in excess,these compounds can become highly toxic.Plants have adopted numerous ways to maintain metal homeostasis while mitigating adverse effects of excess metal ions,including phytochelatin and the metal-chelating proteins metallothioneins(MTs).A family of cysteine(Cys)-rich,intracellular,and low-molecular-weight(4-8 kDa)MTs are proteins found in nearly all phyla including plants,animals,and fungi,and they have the potential to scavenge reactive oxygen species and detoxify toxic metals including copper,cadmium,and zinc.Based on their Cys numbers and residues,MTs have been categorized into three major classes.Class I MTs,which have highly conserved Cys residues,are found in animals,while class II MTs,with less conserved Cys residues,are present in plants and are classified further into four groups.Class III MTs include phytochelatins,a group of enzymatically synthesized Cys-rich proteins.The MTs have been an area of interest for five decades with extensive studies,which have been facilitated by advancements in instrumental techniques,protein science,and molecular biology tools.Here,we reviewed current advances in our understanding of the regulation of MT biosynthesis,their expression,and their potential roles in the alleviation of abiotic stresses(i.e.,drought,salinity,and oxidative stresses)and heavy metal detoxification and homeostasis.
基金the State Key Basic Research and Development Program of China (973 Program, 2007CB108805)
文摘As a member of an important group of lipid soluble antioxidants, tocopherols play a paramount role in the daily diet of humans and animals. Recently, genes required for tocochromanol biosynthesis pathway have been identified and cloned with the help of genomics-based approaches and molecular manipulation in the model organisms： Arabidopsis thaliana and Synechocystis sp. PCC 6803. At the basis of these foundations, genetic manipulation of tocochromanol biosynthesis pathway can give rise to strategies that enhance the level of tocochromanol content or convert the constitution of tocochromanol. In addition, genetic manipulations of the tocochromanol biosynthesis pathway provide help for the study of the function of tocopherol in plant systems. The present article summarizes recent advances and pays special attention to the functions of tocopherol in plants. The roles of tocopherol in the network of reactiv.e oxygen species, antioxidants and phytohormones to maintain redox homeostasis and the functions of tocopherol as a signal molecule in chloroplast-to- nucleus signaling to regulate carbohydrate metabolism are also discussed.
基金the School of Agriculture and Biology, Shanghai Jiao Tong University, China for providing financial support and experimental facilities。
文摘Soils and ecosystems contaminated with cadmium (Cd) threaten human health and adversely affect morphological,physiological,and biochemical parameters of plants.The symbiotic association of endophytic fungi with their host plants is the best strategy to improve various plant characteristics and remediate soils polluted with heavy metal(loid)s (HMs).Being a well-known plant growth-promoting fungus,Piriformospora indica confers resistance against a number of abiotic stresses,including HM stress.This pot experiment explored the potential and ameliorative effects of P.indica on Artemisia annua L.plants treated with different concentrations (0,40,80,and 120 mg kg-1) of Cd.Inoculation with P.indica significantly increased plant performance,especially by enhancing chlorophyll concentration and water potential and by decreasing electrolytic leakage,when compared with un-inoculated plants,despite the high Cd levels.Similarly,P.indica enhanced antioxidant enzyme activities,thereby reducing the drastic effects of Cd in inoculated plants.In addition,P.indica accumulated Cd in the roots of colonized plants,as revealed by atomic absorption spectroscopy,and restricted Cd translocation to aerial parts.Furthermore,P.indica showed in vitro resistance (up to a certain level) to Cd stress;however,fungus growth was inhibited at very high Cd concentrations,proving it an excellent candidate for use as a potential phytoremediator in fields affected by Cd contamination.The transcriptional analysis showed that the signaling genes and artemisinin and flavonoid biosynthetic pathway genes were significantly upregulated in P.indica-co-cultivated plants when compared with un-inoculated plants,suggesting a fine collaboration between primary and secondary metabolisms to modulate resistance capacity and to enhance the phytoremediation capability of A.annua against Cd toxicity.
基金This work was supported by the National Key Research and Development Program of China(No.2016YFD-0800807)the National Nature Science Foundation of China(Nos.31702003 and 31902105)+3 种基金the Young Elite Scientists Sponsorship Program of China Association for Science and Technology(CAST)(No.2017QNRC001)the China Postdoctoral Science Foundation(No.2019 M651505)the“Chenguang Program”of the Shanghai Education Development Foundation and Shanghai Municipal Education Commission,China(No.17CG07)the Shanghai Agriculture Science and Technology Development Project,China(No.2018-4-13).
文摘Trace metals such as manganese(Mn),copper(Cu),zinc(Zn),and iron(Fe)are essential for many biological processes in plant life cycles.However,in excess,they can be toxic and disrupt plant growth processes,which is economically undesirable for crop production.For this reason,processes such as homeostasis and transport control of these trace metals are of constant interest to scientists studying heavily contaminated habitats.Phytoremediation is a promising cleanup technology for soils polluted with heavy metals.However,this technique has some disadvantages,such as the slow growth rate of metal-accumulating plant species,low bioavailability of heavy metals,and long duration of remediation.Microbial-assisted phytoremediation is a promising strategy for hyperaccumulating,detoxifying,or remediating soil contaminants.Arbuscular mycorrhizal fungi(AMF)are found in association with almost all plants,contributing to their healthy performance and providing resistance against environmental stresses.They colonize plant roots and extend their hyphae to the rhizosphere region,assisting in mineral nutrient uptake and regulation of heavy metal acquisition.Endophytic fungi exist in every healthy plant tissue and provide enormous services to their host plants,including growth enhancement by nutrient acquisition,detoxification of heavy metals,secondary metabolite regulation,and enhancement of abiotic/biotic stress tolerance.The aim of the present work is to review the recent literature regarding the role of AMF and endophytic fungi in plant heavy metal tolerance in terms of its regulation in highly contaminated conditions.
文摘Artemisia annua, commonly known as sweet wormwood or Qinghao, is a shrub native to China and has long been used for medicinal purposes. A. annua is now cultivated globally as the only natural source of a potent anti-malarial compound, artemisinin. Here, we report a high-quality draft assembly of the 1.74-gigabase genome of A. annua, which is highly heterozygous, rich in repetitive sequences, and contains 63 226 protein-coding genes, one of the largest numbers among the sequenced plant species. We found that, as one of a few sequenced genomes in the Asteraceae, the A. annua genome contains a large number of genes specific to this large angiosperm clade. Notably, the expansion and functional diversification of genes encoding enzymes involved in terpene biosynthesis are consistent with the evolution of the artemi- sinin biosynthetic pathway. We further revealed by transcriptome profiling that A. annua has evolved the sophisticated transcriptional regulatory networks underlying artemisinin biosynthesis. Based on compre- hensive genomic and transcriptomic analyses we generated transgenic A. annua lines producing high levels of artemisinin, which are now ready for large-scale production and thereby will help meet the chal- lenge of increasing global demand of artemisinin.
基金supported by National High Technology Research and Development Program(2011AA100605)Shanghai Key Discipline Cultivation and Construction Project(Horticulture+1 种基金ZXDF150005)Shanghai Jiao Tong University Agri-Engineering Program(AF1500028)
文摘Artemisinin, also known as qinghaosu, a sesquiterpene endoperoxide lactone isolated from the Chinese medicinal plant Artemisia annua L., is the most effective antimalarial drug which has saved millions of lives.Due to its great antimalarial activity and low content in wild A. annua plants, researches focused on enhancing the artemisin yield in plants became a hotspot. Several families of transcription factors have been reported to participate in regulating the biosynthesis and accumulation of artemisinin.In this review, we summarize recent investigations in these fields, with emphasis on newly identified transcription factors and their functions in artemisinin biosynthesis regulation, and provide new insight for further research.
文摘The voltage-dependent anion channel （VDAC） plays an essential role in the permeability of mltochondrial membrane. In the present study, we isolated a novel VDAC gene （brvdac） based on the assembly of expressed sequence tag sequences from Brassica rapa L. and explored its differential expression patterns In growth, tissues, abiotlc stress, and stress recovery. Results of a tissue-specific expression study in young seedlings Indicated that, of all tissues tested, brvdac expression was the highest in the leaves. Under cold, drought, and salt stresses, brvdac expression showed a transient Increase, and then returned to normal levels when the stress was removed. When plants were exposed to heat shock, there was no Increase in brvdac expression, whereas during recovery a quick and considerable increase in expression was observed. These observations indicate that dissimilar modulations of brvdactranscription may occur when plant cells encounter heat shock and the other three types of stress. In addition, phylogenetic analysis Implied that an earlier duplication of vdac probably occurred before the divergence between monocotyledons and dicotyledons.
基金funded by the National Key R&D Program of China (2019YFC1711100)National Natural Science Foundation of China (32070332)+1 种基金Shanghai Natural Science Foundation in China (20ZR1453800)Shanghai local Science and Technology Development Fund Program guided by the Central Government (YDZX20203100002948)
文摘The important antimalarial drug artemisinin is biosynthesized and stored in Artemisia annua glandular trichomes and the artemisinin content correlates with trichome density;however,the factors affecting trichome development are largely unknown.Here,we demonstrate that the A.annua R2R3 MYB transcription factor TrichomeLess Regulator 1(TLR1)negatively regulates trichome development.In A.annua,TLR1 overexpression lines had 44.7%–64.0%lower trichome density and 11.5%–49.4%lower artemisinin contents and TLR1-RNAi lines had 33%–93.3%higher trichome density and 32.2%–84.0%higher artemisinin contents compared with non-transgenic controls.TLR1 also negatively regulates the expression of anthocyanin biosynthetic pathway genes in A.annua.When heterologously expressed in Arabidopsis thaliana,TLR1 interacts with GLABROUS3a,positive regulator of trichome development,and represses trichome development.Yeast two-hybrid and pull-down assays indicated that TLR1 interacts with the WUSCHEL homeobox(WOX)protein AaWOX1,which interacts with the LEAFY-like transcription factor TLR2.TLR2 overexpression in Arabidopsis and A.annua showed that TLR2 reduces trichome development by reducing gibberellin levels.Furthermore,artemisinin contents were 19%–43%lower in TLR2-overexpressing A.annua plants compared to controls.These data indicate that TLR1 and TLR2 negatively regulate trichome density by lowering gibberellin levels and may enable approaches to enhance artemisinin yields.
基金Sino-UK Science and Technology Collaboration Fund, Shanghai Agriculture Committee，国家自然科学基金
文摘A homeodomain leucine-zipper （HD-Zip） gene BnHB6 （GenBank accession No. AY336103） was isolated from oilseed rape （Brassica napus L.） following drought treatment through rapid amplification of cDNA ends （RACE）. The full-length cDNA of BnHB6 was 1 611 bp and contained a 936-bp open reading frame encoding 311 amino acids. Sequence analysis indicated that BnHB6 belonged to the HD-Zip I subfamily. High-stringency Southern boltting analysis showed that BnHB6 appeared in rape as a single copy but had homologous genes. Semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） analysis revealed that BnHB6 was expressed in several tissues tested under control conditions, but that expression was significantly upregulated in shoots by mannitol, NaCI, cold treatment, anaerobic culture, wounding, H2O2, abscisic acid （ABA）, and salicylic acid （SA） treatments, but not by ultraviolet treatment. Further RT- PCR analysis revealed that BnHB6 was a late-responsive gene, the expression of which was not activated by NaCI, cold treatment, H2O2, ABA, and SA at an early time point （20 min） of treatment in the shoot. However, after a certain period of treatment, the induced expression culminated and then declined until the next peak occurred. Tissue-specific analysis revealed that BnHB6 was expressed at certain levels in the roots, shoots, and flowers, and the roots were found to respond to the osmotic stimuli more rapidly than shoots to increase the expression of BnHB6. The present study implies that BnHB6 plays a positive role as a regulator of biotic and abiotic stresses on growth during seedling establishment.
文摘Catharanthus roseus L. （Madagascar periwinkle） biosynthesizes a diverse array of secondary metabolites including anticancer dimeric alkaloids （vinblastine and vincristine） and antihypertensive alkaloids （ajmalicine and serpentine）. The multi-step terpenoid indole alkaloids （TIAs） biosynthetic pathway in C. roseus is complex and is under strict molecular regulation. Many enzymes and genes involved in the TIAs biosynthesis have been studied in recent decades. Moreover, some regulatory proteins were found recently to control the production of TIAs in C. roseus. Based on mastering the rough scheme of the pathway and cloning the related genes, metabolic engineering of TIAs biosynthesis has been studied in C. roseus aiming at increasing the desired secondary metabolites in the past few years. The present article summarizes recent advances in isolation and characterization of TIAs biosynthesis genes and transcriptional regulators involved in the second metabolic control in C. roseus. Metabolic engineering applications in TIAs pathway via overexpression of these genes and regulators in C. roseus are also discussed.
基金Supported by the Hi-Tech Research and Development(863) Program of China,and the National Natural Science Foundation of China(30500303)
文摘Farnesyl dlphosphate synthase （FPS; EC 2.5.1.10） catalyzes the production of 15-carbon farnesyl dlphosphate which Is a branch-point Intermediate for many terpenoids. This reaction Is considered to be a ratelimiting step In terpenold biosynthesis. Here we report for the first time the cloning of a new full-length cDNA encoding farnesyl dlphosphate synthase from a gymnosperm plant species, Taxus media Rehder, designated as TmFPS1. The full-length cDNA of TmFPS1 （GenBank accession number： AY461811） was 1 464 bp with a 1 056-bp open reading frame encoding a 351-amino acid polypeptlde with a calculated molecular weight of 40.3 kDa and a theoretical pl of 5.07. Biolnformatlc analysis revealed that TmFPS1 contained all five conserved domains of prenyltransferases, and showed homology to other FPSs of plant origin. Phylogenetlc analysis showed that farnesyl dlphosphate synthases can be divided Into two groups： one of prokaryotic origin and the other of eukaryotic origin. TmFPS1 was grouped with FPSs of plant origin. Homologybased structural modeling showed that TmFPS1 had the typical spatial structure of FPS, whose most prominent structural feature Is the arrangement of 13 core helices around a large central cavity In which the catalytic reaction takes place. Our blolnformatic analysis strongly suggests that TmFPS1 is a functional gene. Southern blot analysis revealed that TmFPS1 belongs to a small FPSgene family in T. media. Northern blot analysis indicated that TmFPS1 is expressed in all tested tissues, Including the needles, stems and roots of T. media. Subsequently, functional complementatlon with TmFPS1 in a FPS-deflclent mutant yeast demonstrated that TmFPS1 did encode farnesyl dlphosphate synthase, which rescued the yeast mutant. This study will be helpful In future Investigations aiming at understanding the detailed role of FPS In terpenold biosynthesis flux control at the molecular genetic level.
基金National High-tech R&D Program of China(2011AA100605)Shanghai Key Discipline Cultivation and Construction Project(Horticulture)Shanghai Jiao Tong University Agri-Engineering Program.
文摘Metabolomics has been rapidly developed as an important field in plant sciences and natural products chemistry.As the only natural source for a diversity of monoterpenoid indole alkaloids(MIAs),especially the low-abundance antitumor agents vinblastine and vincristine,Catharanthus roseus is highly valued and has been studied extensively as a model for medicinal plants improvement.Due to multistep enzymatic biosynthesis and complex regulation,genetic modification in the MIA pathway has resulted in complicated changes of both secondary and primary metabolism in C.roseus,affecting not only the MIA pathway but also other pathways.Research at the metabolic level is necessary to increase knowledge on the genetic regulation of the whole metabolic network connected to MIA biosynthesis.Nuclear magnetic resonance(NMR)is a very suitable and powerful complementary technique for the identification and quantification of metabolites in the plant matrix.NMR-based metabolomics has been used in studies of C.roseus for pathway elucidation,understanding stress responses,classification among different cultivars,safety and quality controls of transgenic plants,cross talk between pathways,and diversion of carbon fluxes,with the aim of fully unravelling MIA biosynthesis,its regulation and the function of the alkaloids in the plant from a systems biology point of view.
