Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin i...Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin in the plant. In order to study the regulation of expression of a key enzyme of artemisinin biosynthesis, the promoter region of the key enzyme amorpha-4,11-diene synthase (ADS) was cloned and fused with the β-glucuronidase (GUS) reporter gene. Transgenic plants of A. annua expressing this fusion were generated and studied. Transgenic plants expressing the GUS gene were used to establish the activity of the cloned promoter by a GUS activity staining procedure. GUS under the control of the ADS promoter showed specific expression in glandular trichomes. The activity of the ADS promoter varies temporally and in old tissues essentially no GUS staining could be observed. The expression pattern of GUS and ADS in aerial parts of the transgenic plant was essentially the same indicating that the cis-elements controlling glandular trichome specific expression are included in the cloned promoter. However, some cis-element(s) that control expression in root and old leaf appears to be missing in the cloned promoter. Furthermore, qPCR was used to compare the activity of the wild-type ADS promoter with that of the cloned ADS promoter. The latter promoter showed a considerably lower activity than the wild-type promoter as judged from the levels of GUS and ADS transcripts, respectively, which may be due to the removal of an enhancing cis-element from the ADS promoter. The ADS gene is specifically expressed in stalk and secretory cells of glandular trichomes of A. annua.展开更多
An Amorpha fruticosa cDNA encoding 4 coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism related to lignin forming, was cloned by degenerating oligo primed polymerase chain reaction (PCR) and ...An Amorpha fruticosa cDNA encoding 4 coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism related to lignin forming, was cloned by degenerating oligo primed polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE) PCR. We designed 5′RACE primers based on 4CLA1 fragment which obtained from degenerate PCR. Inverse PCR and nested PCR enabled cloning of the remainder fragments of the gene included 5′ and 3′ end sequence. The ORF encodes a polypeptide of 540 amino acids. The predicted amino acid sequence exhibits significant homology with those of other cloned 4CL genes, contain domains typical of predicted 4CL proteins, in particular a postulated AMP binding site, catalytic domain, and conserved Cys residues.展开更多
Artemisinin, an endoperoxide sesquiterpene lactone has proven effective in treating drug resistant cases of malaria and cancer. Artemisia annua [sweet wormwood] is the sole source for artemisinin production in many co...Artemisinin, an endoperoxide sesquiterpene lactone has proven effective in treating drug resistant cases of malaria and cancer. Artemisia annua [sweet wormwood] is the sole source for artemisinin production in many countries. To counter the low content in leaves and costly chemical synthesis process in India, alternative ways to produce artemisinin have been sought. In current study, we collected A. pallens, A. japonica and A. nilagirica from Western Ghats of Maharashtra, India and analyzed artemisinin content. Samples were extracted from leaves and florets in various extraction conditions and analyzed using different chromatographic techniques. Thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC) analysis showed the presence of compound with endoperoxide linkage in A. nilagirica. High performance liquid chromatography (HPLC) analysis showed the detection of artemisinin in methylene dichloride florets extract of A. japonica, but the concentration was too low [1.3 mg/g dry wt.] for further analyses. Gas chromatography/mass spectrometry (GC/MS) analysis identified structurally important components in the A. nilagirica ethyl acetate extract which explores the biosynthetic pathway of artemisinin from its most important precursor amorpha-4,11-diene. This is the first report of chromatographic screening of these Indian varieties of Artemisia spp. for artemisinin content.展开更多
Artemisia annua is the major natural source of artemisinin,an anti-malarial medicine commonly used worldwide.Here,we present chromosome-level haploid maps for two A.annua strains with different artemisinin contents to...Artemisia annua is the major natural source of artemisinin,an anti-malarial medicine commonly used worldwide.Here,we present chromosome-level haploid maps for two A.annua strains with different artemisinin contents to explore the relationships between genomic organization and artemisinin production.High-fidelity sequencing,optical mapping,and chromatin conformation capture sequencing were used to assemble the heterogeneous and repetitive genome and resolve the haplotypes of A.annua.Approximately 5o,ooo genes were annotated for each haplotype genome,and a triplication event that occurred approximately 58.12million years ago was examined for the first time in this species.A total of 3,903,467-5,193,414 variants(SNPs,indels,and structural variants)were identified in the 1.5-Gb genome during pairwise comparison between haplotypes,consistent with the high heterozygosity of this species.Genomic analyses revealed a correlation between artemisinin concents and the copy number of amorpha-4,11-dienes ynthasegenes.This correlation was further confirmed by resequencing of 36A.annua samples with varied artemisinin contents.Circular consensus sequencing of transcripts facilitated the detection of paralog expression.Collectively,our study provides chromosome-level allele-aware genome assemblies for two A.annua strains and new insights into the biosynthesis of artemisinin and its regulation,which will contribute to conquering malaria worldwide.展开更多
文摘Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin in the plant. In order to study the regulation of expression of a key enzyme of artemisinin biosynthesis, the promoter region of the key enzyme amorpha-4,11-diene synthase (ADS) was cloned and fused with the β-glucuronidase (GUS) reporter gene. Transgenic plants of A. annua expressing this fusion were generated and studied. Transgenic plants expressing the GUS gene were used to establish the activity of the cloned promoter by a GUS activity staining procedure. GUS under the control of the ADS promoter showed specific expression in glandular trichomes. The activity of the ADS promoter varies temporally and in old tissues essentially no GUS staining could be observed. The expression pattern of GUS and ADS in aerial parts of the transgenic plant was essentially the same indicating that the cis-elements controlling glandular trichome specific expression are included in the cloned promoter. However, some cis-element(s) that control expression in root and old leaf appears to be missing in the cloned promoter. Furthermore, qPCR was used to compare the activity of the wild-type ADS promoter with that of the cloned ADS promoter. The latter promoter showed a considerably lower activity than the wild-type promoter as judged from the levels of GUS and ADS transcripts, respectively, which may be due to the removal of an enhancing cis-element from the ADS promoter. The ADS gene is specifically expressed in stalk and secretory cells of glandular trichomes of A. annua.
文摘An Amorpha fruticosa cDNA encoding 4 coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism related to lignin forming, was cloned by degenerating oligo primed polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE) PCR. We designed 5′RACE primers based on 4CLA1 fragment which obtained from degenerate PCR. Inverse PCR and nested PCR enabled cloning of the remainder fragments of the gene included 5′ and 3′ end sequence. The ORF encodes a polypeptide of 540 amino acids. The predicted amino acid sequence exhibits significant homology with those of other cloned 4CL genes, contain domains typical of predicted 4CL proteins, in particular a postulated AMP binding site, catalytic domain, and conserved Cys residues.
文摘Artemisinin, an endoperoxide sesquiterpene lactone has proven effective in treating drug resistant cases of malaria and cancer. Artemisia annua [sweet wormwood] is the sole source for artemisinin production in many countries. To counter the low content in leaves and costly chemical synthesis process in India, alternative ways to produce artemisinin have been sought. In current study, we collected A. pallens, A. japonica and A. nilagirica from Western Ghats of Maharashtra, India and analyzed artemisinin content. Samples were extracted from leaves and florets in various extraction conditions and analyzed using different chromatographic techniques. Thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC) analysis showed the presence of compound with endoperoxide linkage in A. nilagirica. High performance liquid chromatography (HPLC) analysis showed the detection of artemisinin in methylene dichloride florets extract of A. japonica, but the concentration was too low [1.3 mg/g dry wt.] for further analyses. Gas chromatography/mass spectrometry (GC/MS) analysis identified structurally important components in the A. nilagirica ethyl acetate extract which explores the biosynthetic pathway of artemisinin from its most important precursor amorpha-4,11-diene. This is the first report of chromatographic screening of these Indian varieties of Artemisia spp. for artemisinin content.
基金This work was supported by the National Key R&D Program of China(2019YFC1711100)the National Natural Science Foundation of China(U1812403-1,81641002)+1 种基金the Fundamental Research Funds forthe Central Public Welfare Research Institutes(ZZ13-YQ-047,ZZ13-YQ-102)the National Major Science and Technology Projects(2017ZX09101002-003-001,2019ZX09201005-006-001,2019ZX09201005-002-002).
文摘Artemisia annua is the major natural source of artemisinin,an anti-malarial medicine commonly used worldwide.Here,we present chromosome-level haploid maps for two A.annua strains with different artemisinin contents to explore the relationships between genomic organization and artemisinin production.High-fidelity sequencing,optical mapping,and chromatin conformation capture sequencing were used to assemble the heterogeneous and repetitive genome and resolve the haplotypes of A.annua.Approximately 5o,ooo genes were annotated for each haplotype genome,and a triplication event that occurred approximately 58.12million years ago was examined for the first time in this species.A total of 3,903,467-5,193,414 variants(SNPs,indels,and structural variants)were identified in the 1.5-Gb genome during pairwise comparison between haplotypes,consistent with the high heterozygosity of this species.Genomic analyses revealed a correlation between artemisinin concents and the copy number of amorpha-4,11-dienes ynthasegenes.This correlation was further confirmed by resequencing of 36A.annua samples with varied artemisinin contents.Circular consensus sequencing of transcripts facilitated the detection of paralog expression.Collectively,our study provides chromosome-level allele-aware genome assemblies for two A.annua strains and new insights into the biosynthesis of artemisinin and its regulation,which will contribute to conquering malaria worldwide.
