OBJECTIVETo study the distribution frequency and characteristics of nucleotide repeat of 94 923 ESTs for developing microsatellite primers, providing a theoretical basis and technical support for appropriate conservation and application of sweet wormwood (Artemisia annua).

METHODEST-SSR detection was performed using Perl program MISA. Gene Ontology (GO) annotations were formatted for input into the GOSlim program and the output was parsed to count the occurrence of each GO category. Primer 3 software was used to design 18 pairs primers, amplified products were separated on a 6% denaturing polyacrylamide gel using silver staining.

RESULTBy searching with Active Perl, totally 2 110 SSRs were detected, accounting for 8.6%. The frequency of occurrence of dinucleotide and trinucleotide was 28% and 50.4%, respectively. The most common repeat motifs of trinucleotide were ACC/GGT, accounting for 9.8%. Three hundred and twelve SSR-ESTs were annotated using GO terms. The suitable PCR system of 15 pairs primers was established, and revealed microsatellite polymorphism in 36 individuals.

CONCLUSIONThere are a variety of motifs at EST-SSR locus in sweet wormwood, and more effective amplification and polymorphism in 18 pairs detected primers. Therefore, EST resource is an effective and feasible approach to develop SSR markers, and EST-SSRs will a powerful tool for studies of sweet wormwood genetic resources.

Artemisia annua ; genetics ; Expressed Sequence Tags ; Microsatellite Repeats ; Polymorphism, Genetic

OBJECTIVETo study the relative expression of the genes involved in artemisinin biosynthesis in different tissues including roots, stems, leaves and flowers of Artemisia annua, and establish the relationship between gene expression and artemisinin accumulation, eventually leading to discover the mainly effective genes involved in artemisinin biosynthesis.

METHODThe 7 functional genes involved in artemisinin biosynthesis were detected at the level of expression by using qRT-PCR, and simultaneously the content of artemisinin in the 4 investigated tissues was detected in parallel.

RESULTThe 3 genes including HMGR, DXR and FPS which were involved in the upstream pathway of artemisinin biosynthesis showed the highest expression levels in flowers, and the 4 functional genes including ADS, CYP71AV1, CPR and AAR which were involved in the artemisinin-specific biosynthetic pathway were found to be expressed in all the 4 detected tissues. The highest expression level of ADS was found in leaves, then followed by flowers, and the lowest expression level of ADS was found in roots and stems. CYP71AV1 had highest expression level in flowers and lowest in leaves. CPR showed highest expression level in flowers, and AAR had lower expression levels in the other 3 artemisinin-specific pathway genes in all the tissues. The highest content of artemisinin was found in leaves (0.343 mg x g(-1)), then followed by flowers (0.152 mg x g(-1)), roots (0.062 mg x g(-1)) and stems (0.060 mg x g(-1)).

CONCLUSIONIn the biosynthesis of artemisinin, the upstream genes including HMGR from the MVA pathway, DXR from the MEP pathway and the checkpoint gene FPS were much more active in flowers, and this suggested that flowers might be the tissues of artemisinin precursor biosynthesis, and further DXR contributed more to artemisinin biosynthesis. The positive correlation of ADS expression and artemisinin content in tissues demonstrated that ADS played a very important role in artemisinin biosynthesis, which was the ideal target for engineering the artemisinin biosynthetic pathway. In summary, the functional genes involved in artemisinin biosynthesis do not express at the same level but synergistically.

Artemisia annua ; chemistry ; genetics ; metabolism ; Artemisinins ; metabolism ; Plant Proteins ; genetics ; metabolism ; Polymerase Chain Reaction

As a natural plant source of artemisinin,a first-line drug against malaria,Artemisia annua directly affects the extraction process of artemisinin and the source of artemisinin. At present,traditional breeding methods combined with tissue culture are often used to breed high-yield artemisinin-containing new varieties of A. annua. However,the breeding method has the disadvantages of low efficiency and continuous selection. In this study,heavy ion beam irradiation technology was used to observe the specific germplasm resources of A. annua,and the morphological characteristics,agronomic traits and artemisinin content were used as indicators to observe the selection materials and materials. The cultivated new varieties were compared with trials and regional trials. In addition,the new variety of A. annua was identified by SRAP molecular marker technology. The results showed that the new variety of A. annua, " Kehao No.1",had an average yield of 235. 0 kg of dry leaf per mu,which was more than 20% higher than that of the control. Especially,the average artemisinin content was 2. 0%,which was 45% higher than that of the control,and the " Kehao No.1" has high anti-white powder disease,high-yield and high-quality new varieties. Therefore,mutagenic breeding of heavy ion beam irradiation can significantly improve the yield and artemisinin content of the " Kehao No. 1" and it has a good promotion value.
Artemisia annua/genetics* ; Artemisinins/analysis* ; Heavy Ions ; Mutagenesis ; Phenotype ; Plant Breeding ; Plants, Medicinal/genetics*

OBJECTIVETo breed and spread a new cultivar of Artemisia annua.

METHODThe excellent germplasm resources of A. annua in the main production area of Artemisia were collected, and the improved germplasm were screened, the content of artemisinin was determined, and yield per plant was measured. The systematically maternal line and seed production techniques of mass selection were applied combined with the variety test, variety regional test trials and production trials for breeding and spreading the new cultivars of artemisia.

RESULTThe popularization and experiment illustrated the production of the new species reached 3 000 kg x hm(-2), compared with wild A. annua it increased 10% -14%. The content of artemisinin reached more than 1%, increased more than 0.2%.

CONCLUSIONIt is proved that the systematically maternal line and seed production techniques of mass selection can significantly improve the quality of A. annua and it is an acceptable way to cultivate new variety. By production verification, it is practicable and high technical and economic benefits to popularize the new cultivar "Yu-Qing No. 1" of A. annua.

Antimalarials ; analysis ; pharmacology ; Artemisia annua ; chemistry ; genetics ; growth & development ; Artemisinins ; analysis ; pharmacology ; Breeding ; methods

OBJECTIVETo evaluate the diversity of germplasm resources of Artemisia annua and provide the basis for improving utilization of germplasm resources, the agronomic traits of germplasm resources of A. annua were studied in Yun-Gui plateau.

METHODThe agronomic traits of 67 A. annua germplasm resources were measured by the visual observation and measurement methods. And the germplasm resources were clustered using flexible-beta method to analysis their genetic background.

RESULTThe result showed that 67 germplasm resources had a relatively wide variation on the 22 agronomic traits. Among 22 agronomic traits, the dry weight of branch had the greatest coefficient of variation, which was 53. 63, and the next were the dry weight of leaf, total plant weight, the length of pinnules and the length of leaflet, which were 42.74, 41.61, 39.54 and 39.22 respectively. The smallest coefficient of variation was the leaf corlor. Based the result of cluster analysis, these 67 germplasm resources were classed into 5 groups, and each group had its respective character. The first group showed early-maturing resources, dwarf stalk, slender rod, long bipinnata, high leaf-stem ratio and moderate leaf weight The third group showed late-maturing resources, tall and thick stalk, much-branch, bushy accessory pinna, high leaf weight and yield. The fifth group showed very late-maturing resources, strong lateral shoot, high leaf yield.

CONCLUSIONThere were significant genetic difference and diversity in the germplasm resources of A. annua. The result of cluster analysis showed that the resources of group 1, group 3 and group 5 were suitable as breeding material of A. annua.

Artemisia annua ; classification ; genetics ; growth & development ; Biodiversity ; Biomass ; China ; Cluster Analysis

OBJECTIVETo try to find the ways to enhance the expression of ADS gene encoding amorpha-4,11-diene synthase, a key enzyme in artemisinin biosynthesis pathway catalyzing the formation of amorpha-4,11-diene from farnesyl diphosphate, and accelerate the artemisinin synthesis, the promoter of ADS was isolated and characterized.

METHOD5' untranslated regions of ADS were isolated from Artemisia annua with PCR. For functional characterization, the isolated fragment was fused with GUS reporter gene and introduced into Nicotiana tabacum by Agrobacterium-mediated transformation. The GUS expression regulated by 5' untranslated regions of ADS in transgenic N. tabacum under the normal or stressed conditions were detected by histochemical staining and quantitative spectrophotometry assay.

RESULTThe 2 448 bp DNA fragment upstream of ADS coding sequence was isolated from A. annua and introduced into N. tabacum. Histochemical staining showed that the isolated fragment conferred stable GUS expression in transgenic plants. The quantitative results showed that the GUS activity in transgenic tobacco plants treated by low-temperature (4 degrees C) and ultraviolet irradiation were 1. 6 and 2.2 folds higher than that in the controls.

CONCLUSIONIt was suggested that the isolated fragment had promoter activity and maybe responsive to adverse environmental stresses.

5' Untranslated Regions ; genetics ; Alkyl and Aryl Transferases ; genetics ; metabolism ; Artemisia annua ; enzymology ; genetics ; Gene Expression Regulation, Plant ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics

To reveal the genetic diversity and genetic structure in Artemisia annua varieties (strains) populations, we detected the genetic polymorphism within and among eight varieties (strains) populations (192 individuals) by the approach of Start Codon Targeted Polymorphism (SCoT). The associated genetic parameters were calculated by POPGENE1.31 and the relationship was constructed based on UPGMA method. The results showed that, using 20 screened primers, a total of 145 bands were produced, of which 122 were polymorphic loci. At species level, there was a high level of genetic diversity among eight varieties (strains) populations (PPB = 84.1% ,H = 0.217 3 and H(sp) = 0.341 9). However, at the variety (strains) population level, genetic diversity was lower, the average of genetic parameters was PPB = 41.9%, H = 0.121 5, H(pop) = 0.186 8. The Nei's genetic differentiation coefficient was 0.441 0, indicate that most of the genetic variation in this species existed within the variety populations. The gene flow (N(m) = 0.633 9) was less among populations, indicating that the degree of genetic differentiation was higher. Genetic similarity coefficient were changed from 0.755 1 to 0.985 7. By clustering analysis, eight varieties (strains) were clustered into two major categories and it was also showed the same or similar genetic background varieties (strains) have a tendency to gather in the same group. Results suggest that, in variety breeding, breeders should strengthen the exchange of bred germplasm and increase mutual penetration of excellent genes, which would broaden the genetic base of A. annua.
Artemisia annua ; classification ; genetics ; Codon, Initiator ; genetics ; Genetic Markers ; genetics ; Genetic Structures ; Genetic Variation ; Genetics, Population ; methods ; Phylogeny ; Polymorphism, Genetic ; Species Specificity

Artemisinin, a new and a very potent antimalarial drug, is produced by the Chinese medicinal herb Artemisia annua L. It is a sesquiterpene lactone with an endoperoxide bridge and is active against chloroquine resistant forms of Plasmodium falciparum. The relatively low yield (0.01% - 0.6%) of artemisinin in A. annua is a serious limitation to the commercialization of the drug. Therefore, a through understanding of the biosynthetic pathway and the characterization of the involved enzymes are important for the biology production of artemisinin. This review is focused on the recent progress in the molecular regulation of artemisinin biosynthesis from the following aspects: the biosynthetic pathway of artemisinin, the key enzymes involved in artemisinin biosynthesis, and the molecular regulation of artemisinin biosynthesis. The biosynthetic pathway of artemisinin belongs to the isoprenoid metabolite pathway, the key enzymes involved in the biosynthesis of artemisinin include: 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), farnesyl diphosphate synthase (FDPS), and amorpha-4, 11-diene synthase, of which amorpha-4, 11-diene synthase catalyzes the cyclisation of the ubiquitous precursor farnesyl diphosphate to the highly specific olefinic sesquiter-pene skeletons and has been postulated as the regulatory step in the biosynthesis of artemisinin. Recently the gene encoding of the amorpha-4, 11-diene synthase has been cloned and the functional expressions have been studied by several research teams, therefore, the breakthroughs in production of artemisinin could hopefully be achieved by metabolic engineering of the plant, in particular, by over-expressing enzyme(s) catalyzing the rate limiting step(s) of artemisinin biosynthesis or by inhibiting the enzyme(s) of other pathway competing for its precursors. Besides, the effects of the heterogenesis isoprenoid pathway related genes on artemisinin biosynthesis of the transformed plants were also discussed.
Alkyl and Aryl Transferases ; genetics ; metabolism ; Antimalarials ; metabolism ; Artemisia annua ; enzymology ; genetics ; metabolism ; Artemisinins ; metabolism ; Biotechnology ; methods ; Models, Biological ; Signal Transduction ; genetics ; physiology

In this study, Actin, 18S rRNA, PAL, GAPDH and CPR of Artemisia annua were selected as candidate reference genes, and their gene-specific primers for real-time PCR were designed, then geNorm, NormFinder, BestKeeper, Delta CT and RefFinder were used to evaluate their expression stability in the leaves of A. annua under treatment of different concentrations of Cd, with the purpose of finding a reliable reference gene to ensure the reliability of gene-expression analysis. The results showed that there were some significant differences among the candidate reference genes under different treatments and the order of expression stability of candidate reference gene was Actin > 18S rRNA > PAL > GAPDH > CPR. These results suggested that Actin, 18S rRNA and PAL could be used as ideal reference genes of gene expression analysis in A. annua and multiple internal control genes were adopted for results calibration. In addition, differences in expression stability of candidate reference genes in the leaves of A. annua under the same concentrations of Cd were observed, which suggested that the screening of candidate reference genes was needed even under the same treatment. To our best knowledge, this study for the first time provided the ideal reference genes under Cd treatment in the leaves of A. annua and offered reference for the gene expression analysis of A. annua under other conditions.
Artemisia annua ; drug effects ; genetics ; metabolism ; Cadmium ; pharmacology ; Plant Leaves ; drug effects ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; methods ; standards ; Reference Standards

Amorpha-4,11-diene synthase (ADS) can convert farnesyl pyrophosphate (FPP) to amorpha-4, 11-diene, a precursor of artemisinin. ADS plays an important role in the biosynthesis of artemisinin. This review summarizes the molecular biology and metabolic engineering study of ADS in recent years. The genomic DNA and its cDNA sequences of amorpha-4, 11-diene synthase were cloned from Artemisia annua L. The cDNA encoding amorpha-4, 11-diene synthase contains a 1 641 bp open reading frame coding for 546 amino acids. ADS shows a broad pH optimum and an absolute requirement for divalent metal ions as cofactors. The specificity of ADS to the substrates and products is not high and the formation of amorpha-4, 11-diene by ADS from FPP is achieved by an initial 1, 6-closure with subsequent 1, 10-closure. The ADS cDNA cloned from Artemisia annua L, or totally synthesized by PCR, was introduced into different hosts including E. coli, S. cerevisiae, Nicotiana tabacum L. Arabidopsis thaliana and A. nidulans resulting in varied engineering microorganisms and cells producing amorpha-4, 11-diene. The way to improve the production of amorpha-4, 11-diene was investigated by two strategies such as improving the supply of substrate and directing FPP flux to amorpha-4, 11-diene production from competing pathways.
Alkyl and Aryl Transferases ; biosynthesis ; genetics ; Amino Acid Sequence ; Antimalarials ; metabolism ; Arabidopsis ; enzymology ; genetics ; Artemisia annua ; enzymology ; genetics ; Artemisinins ; metabolism ; Aspergillus ; genetics ; metabolism ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Metabolic Engineering ; Saccharomyces cerevisiae ; genetics ; metabolism ; Tobacco ; enzymology ; genetics