[Quantitative Comparison of Methods to Assess the Airborne Particulate Matter Retention Capacity of Leaves].

The objective of this study was to explore the differences of five methods for evaluating the PM retention capacity of leaves based on the same experimental materials and leaf area measurement method and to summarize the advantages and disadvantages of each method. In this study, four tree species (Pinus tabuliformis, Platycladus orientalis, Ginkgo biloba, and Platanus occidentalis), which are common in Beijing and have greatly different leaf characteristics, were selected as the research objects. The mass subtraction method (MS), the membrane filter method (MF), the aerosol regenerator method(AR), the scanning electron method (SEM), and the elution weighing method coupled with a particle size analysis based on ultrasonic cleaning (ultrasonic-EWPA) were used to evaluate the PM retention capabilities and characteristics of the leaves of the four tree species. The total time needed and the total cost were measured simultaneously during the evaluation process. The results showed that although the values of PM retention efficiency obtained by different methods were quite different, the ranks of the efficiency of four tree species obtained by different methods were the same or partially the same. Additionally, the results obtained by the methods with the same or similar principles were more overlapped (AR and SEM had the most overlapped results). In addition, 89% of the species ranks of the same index obtained by each method were P. orientalis > P. tabuliformis > P. occidentalis > G. biloba, and the remaining 11% were P. occidentalis > G. biloba > P. orientalis > P. tabuliformis. Among the five methods, ultrasonic-EWPA was the one with the largest number of indexes and the highest cost, and MS was the one with the least number of indexes and the lowest cost. The one that needed most time was MF, while the one that needed the least time was AR. ultrasonic-EWPA and SEM are high input and high output methods. That is to say, although they needed more time and cost, they can prove more information; however, MS was opposite, which resulted in less information but lower time and cost needed. So, it is suitable for roughly evaluating the total PM retention capacities of trees; MF had a medium amount of information, low cost, but required too much time, which needs to be weighed and balanced before selecting this method. The AR method had strict requirements for equipment and parameters and should be used with caution. The results of this study can provide a comprehensive and detailed scientific basis for researchers to choose specific methods in the future.

[Effects of water and nitrogen coupling under furrow irrigation on tree growth, absorption and utilization of water and nitrogen of Populus tomentosa]. / 沟灌水氮耦合对毛白杨林木生长及水氮吸收利用的影响.

We explored the coupling effects of water and nitrogen in furrow irrigation on the growth and absorption and utilization of water and nitrogen in young poplar trees (triploid Populus tomentosa), under three irrigation levels (W20, W33, W45; when the soil water potential of 40 cm under the ditch reaches -20, -33 and -45 kPa respectively, irrigate), four N application levels (N120, N190, N260 and N0; the fertilization amount was 120, 190, 260 and 0 kg·hm-2·a-1), and natural conditions (CK). Based on the growth status of trees, the optimal combination of irrigation level and nitrogen application rate under furrow irrigation conditions was determined. The results showed that W20N120 (high water and low fertilizer; soil water potential threshold for initiating irrigation was -20 kPa and N application rate was 120 kg·hm-2·a-1) had the strongest effect on the stand productivity, with a value of 33.37 m3·hm-2·a-1. The significant coupling effect of water and N was detected only for tree height and total individual biomass. The increase of both irrigation amount and N application rate could increase the amount of N uptake, being mainly affected by the latter. The total amount of N uptake was the highest in the W20N260 treatment and reached 112.17 kg·hm-2·a-1, being 74.0% higher than that in CK. Among all the treatments, N uptake efficiency and N fertilizer partial productivity of W20N120 were the highest and significantly higher than those of the other treatments. The N uptake efficiency of the whole plant, aboveground part, and belowground part reached 36.8%, 28.5% and 6.4% in the W20N120 treatment, and its total N partial productivity reached 221.4 kg·kg-1. The effect of irrigation amount under different water-nitrogen coupling treatments on the irrigation water use efficiency was significant. Among them, irrigation water use efficiency in W45N260 was the highest and reached 13.66 g·kg-1. W20N120 had the highest water uptake amount and efficiency, which were 13268.28 t·hm-2 and 129.4%, respectively. To achieve great benefits, adequate irrigation (-20 kPa) and relatively low N application rate (120 kg·hm-2·a-1) should be selected during the young growth of the triploid P. tomentosa.

[Quantitative evaluation for separation of water-soluble and water-insoluble particulate matter on leaf surface of tree species: Taking five tree species as examples.] / 植物叶片表面水溶与非水溶性颗粒物滞纳量分离定量评估­­ä»¥5ç§æ ‘ç§ä¸ºä¾‹.

To accurately and quantitatively evaluate the mass and particle size distribution of water-soluble and water-insoluble particulate matters (PM) on the surface of tree leaves, which would help to improve the accuracy of quantitative assessment of the retention ability of urban trees to atmospheric particles, we collected leaf samples from three broadleaved tree species [Ginkgo (Ginkgo biloba), Chinese scholar tree (Sophora japonica) and weeping willow (Salix babylonica)] and two conifer species [Chinese pine (Pinus tabuliformis) and China savin (Sabina chinensis)] 14 d after the rain (rainfall>15 mm). The PMs retained on leaves were collected by a succeeding procedure of washing + brushing (WC+BC) and ultrasonic cleaning (UC). Then, the extracts at each step were divided into water-soluble and water-insoluble PMs through centrifuge. The mass of water-soluble and water-insoluble particles were dry weighted. Then, the water-soluble and water-insoluble particles were dissolved by anhydrous ethanol and deionized water to measure the particle size distribution. The mass of water-soluble and water-insoluble particles with different particle sizes was calculated. Results showed that the mass (proportion) of water-soluble PMs retained on leaf surfaces of broad-leaved and conifer species were 480.61 (52.3%) and 438.91 (47.7%) mg·m-2, respectively, while that for water-insoluble PMs were 97.93 (12.0%) and 715.84 (88.0%) mg·m-2, respectively. The particle size distribution of water-soluble particles on the leaves of the five tree species showed the unimodal curve with mean size of 40.36 µm. Water-insoluble particles on leaves showed multimodal distribution, with mean size of 105.65 µm. S. japonica and G. biloba had higher PM retention ability in regions suffering with more water-soluble PM pollution, while S. chinensis had higher retention ability to water-insoluble PMs.

[Effects of fertilization method and nitrogen application rate on soil nitrogen vertical migration in a Populus xeuramericana cv. 'Guariento' plantation].

A field experiment was conducted to investigate the effects of fertilization methods, i.e., drip (DF) and furrow fertilization (GF), and nitrogen (N) application rates (25, 50, 75 g N · plant(-1) · time(-1)) on the dynamics of soil N vertical migration in a Populus x euramericana cv. 'Guariento' plantation. The results showed that soil NH4(+)-N and NO3(-)-N contents decreased with the increasing soil depth under different fertilization methods and N application rates. In the DF treatment, soil NH4(+)-N and NO3(-)-N were mainly concentrated in the 0-40 cm soil layer, and their contents ascended firstly and then descended, reaching their maximum values at the 5th day (211.1 mg · kg(-1)) and 10th day (128.8 mg · kg(-1)) after fertilization, respectively. In the GF treatment, soil NH4(+)-N and NO3(-)-N were mainly concentrated in the 0-20 cm layer, and the content of soil NO3(-)-N rose gradually and reached its maximum at the 20th day (175.7 mg · kg(-1)) after fertilization, while the NH4(+)-N content did not change significantly after fertilization. Overall, N fertilizer had an effect within 20 days in the DF treatment, and more than 20 days in the GF treatment. In the DF treatment, the content and migration depth of soil NH4(+)-N and NO3(-)-N increased with the N application rate. In the GF treatment, the NO3(-)-N content increased with the N application rate, but the NH4(+)-N content was not influenced. Under the DF treatment, the hydrolysis rate, nitrification rate and migration depth of urea were higher or larger than that under the GF treatment, and more N accumulated in deep soil as the N application rate increased. Considering the distribution characteristics of fine roots and soil N, DF would be a better fertilization method in P. xeuramericana cv. 'Guariento' plantation, since it could supply N to larger distribution area of fine roots. When the N application rate was 50 g · tree(-1) each time, nitrogen mainly distributed in the zone of fine roots and had no risk of deep leaching, consequently improving the fertilizer utilization efficiency.

[Exploration of a quantitative methodology to characterize the retention of PM2.5 and other atmospheric particulate matter by plant leaves: taking Populus tomentosa as an example].

Taking Populus tomentosa as an example, a methodology called elution-weighing-particle size-analysis (EWPA) was proposed to evaluate quantitatively the ability of retaining fine particulate matter (PM2.5, diameter d ≤ 2.5 µm) and atmospheric particulate matter by plant leaves using laser particle size analyzer and balance. This method achieved a direct, accurate measurement with superior operability about the quality and particle size distribution of atmospheric particulate matter retained by plant leaves. First, a pre-experiment was taken to test the stability of the method. After cleaning, centrifugation and drying, the particulate matter was collected and weighed, and then its particle size distribution was analyzed by laser particle size analyzer. Finally, the mass of particulate matter retained by unit area of leaf and stand was translated from the leaf area and leaf area index. This method was applied to a P. tomentosa stand which had not experienced rain for 27 days in Beijing Olympic Forest Park. The results showed that the average particle size of the atmospheric particulate matter retained by P. tomentosa was 17.8 µm, and the volume percentages of the retained PM2.5, inhalable particulate matter (PM10, d ≤ 10 µm) and total suspended particle (TSP, d ≤ 100 µm) were 13.7%, 47.2%, and 99.9%, respectively. The masses of PM2.5, PM10, TSP and total particulate matter were 8.88 x 10(-6), 30.6 x 10(-6), 64.7 x 10(-6) and 64.8 x 10(-6) g x cm(-2) respectively. The retention quantities of PM2.5, PM10, TSP and total particulate matter by the P. tomentosa stand were 0.963, 3.32, 7.01 and 7.02 kg x hm(-2), respectively.

Early compliance and efficacy of sublingual immunotherapy in patients with allergic rhinitis / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#The study aimed to investigate the efficacy and adverse effects of sublingual immunotherapy (SLIT) of dust mite drops to allergic rhinitis with mite allergy. The compliance and satisfaction of SLIT were also assessed.@*METHOD@#One hundred and three patients of allergic rhinitis sensitive to dust mites were treated with SLIT for 6 months or more. The symptom questionnaire,including items on rhinorrhea, sneezing, nasal obstruction, itchy nose, olfactory disturbance, eye discomfort and sleep disturbance were obtained before and 6 months after SLIT. The patients' satisfaction and adverse effects were also investigated.@*RESULT@#Seventy-five of the 103 patients insist on SLIT for more than 6 months and completed the questionnaire. The duration of receiving SLIT was 9.8 months on average (range from 6 to 13 months). The satisfaction rate was 89.3%. The drop-out rate of SLIT was 31.0%.@*CONCLUSION@#The subjective symptoms were improved with SLIT in patients with allergic rhinitis sensitive to dust mites. The drop out rate was high despite of the symptomatic improvement.

[Simulation of soil water dynamics in triploid Populus tomentosa root zone under subsurface drip irrigation].

Based on the observed data of triploid Populus tomentosa root distribution, a one-dimensional root water uptake model was proposed. Taking the root water uptake into account, the soil water dynamics in triploid P. tomentosa root zone under subsurface drip irrigation was simulated by using HYDRUS model, and the results were validated with field experiment. Besides, the HYDRUS model was used to study the effects of various irrigation technique parameters on soil wetting patterns. The RMAE for the simulated soil water content by the end of irrigation and approximately 24 h later was 7.8% and 6.0%, and the RMSE was 0.036 and 0.026 cm3 x cm(-3), respectively, illustrating that the HYDRUS model performed well in simulating the short-term soil water dynamics in triploid P. tomentosa root zone under drip irrigation, and the root water uptake model was reasonable. Comparing with 2 and 4 L x h(-1) of drip discharge and continuous irrigation, both the 1 L x h(-1) of drip discharge and the pulsed irrigation with water applied intermittently in 30 min periods could increase the volume of wetted soil and reduce deep percolation. It was concluded that the combination of 1 L x h(-1) of drip discharge and pulsed irrigation should be the first choice when applying drip irrigation to triploid P. tomentosa root zone at the experiment site.

Artemisinin biosynthesis enhancement in transgenic Artemisia annua plants by downregulation of the ß-caryophyllene synthase gene.

Artemisinin is an effective antimalarial drug isolated from the medicinal plant Artemisia annua L. Due to its increasing market demand and the low yield in A. annua, there is a great interest in increasing its production. In this paper, in an attempt to increase artemisinin content of A. ANNUA by suppressing the expression of ß-caryophyllene synthase, a sesquiterpene synthase competing as a precursor of artemisinin, the antisense fragment (750 bp) of ß-caryophyllene synthase cDNA was inserted into the plant expression vector pBI121 and introduced into A. annua by Agrobacterium-mediated transformation. PCR and Southern hybridization confirmed the stable integration of multiple copies of the transgene in 5 different transgenic lines of A. annua. Reverse transcription PCR showed that the expression of endogenous CPS in the transgenic lines was significantly lower than that in the wild-type control A. annua plants, and ß-caryophyllene content decreased sharply in the transgenic lines in comparison to the control. The artemisinin content of one of the transgenic lines showed an increase of 54.9 % compared with the wild-type control. The present study demonstrated that the inhibition pathway in the precursor competition for artemisinin biosynthesis by anti-sense technology is an effective means of increasing the artemisinin content of A. annua plants.

Salicylic acid activates artemisinin biosynthesis in Artemisia annua 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.

Identification of a Polygonum cuspidatum three-intron gene encoding a type III polyketide synthase producing both naringenin and p-hydroxybenzalacetone.

Benzalacetone synthase (BAS) is a member of the plant-specific type III PKS superfamily that catalyzes a one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce p-hydroxybenzalacetone. In our recent work (Ma et al. in Planta 229(3):457-469, 2008), a three-intron type III PKS gene (PcPKS2) was isolated from Polygonum cuspidatum Sieb. et Zucc. Phylogenetic and functional analyses revealed this recombinant PcPKS2 to be a BAS. In this study, another three-intron type III PKS gene (PcPKS1) and its corresponding cDNA were isolated from P. cuspidatum. Sequence and phylogenetic analyses demonstrated that PcPKS1 is a chalcone sythase (CHS). However, functional and enzymatic analyses showed that recombinant PcPKS1 is a bifunctional enzyme with both, CHS and BAS activity. DNA gel blot analysis indicated that there are two to four CHS copies in the P. cuspidatum genome. RNA gel blot analysis revealed that PcPKS1 is highly expressed in the rhizomes and in young leaves, but not in the roots of the plant. PcPKS1 transcripts in leaves were inducible by pathogen infection and wounding. BAS is thought to play a crucial role in the construction of the C(6)-C(4) moiety found in a variety of phenylbutanoids, yet so far phenylbutanoids have not been isolated from P. cuspidatum. However, since PcPKS1 and PcPKS2 (Ma et al. in Planta 229(3):457-469, 2008) have been identified in P. cuspidatum, it is possible that such compounds are also produced in that plant, albeit in low concentrations.

A novel type III polyketide synthase encoded by a three-intron gene from Polygonum cuspidatum.

A type III polyketide synthase cDNA and the corresponding gene (PcPKS2) were cloned from Polygonum cuspidatum Sieb. et Zucc. Sequencing results showed that the ORF of PcPKS2 was interrupted by three introns, which was an unexpected finding because all type III PKS genes studied so far contained only one intron at a conserved site in flowering plants, except for an Antirrhinum majus chalcone synthase gene. Besides the unusual gene structure, PcPKS2 showed some interesting characteristics: (1) the CHS "gatekeepers" Phe215 and Phe265 are uniquely replaced by Leu and Cys, respectively; (2) recombinant PcPKS2 overexpressed in Escherichia coli efficiently afforded 4-coumaroyltriacetic acid lactone (CTAL) as a major product along with bis-noryangonin (BNY) and p-hydroxybenzalacetone at low pH; however, it effectively yielded p-hydroxybenzalacetone as a dominant product along with CTAL and BNY at high pH. Beside p-hydroxybenzalacetone, CTAL and BNY, a trace amount of naringenin chalcone could be detected in assays at different pH. Furthermore, 4-coumaroyl-CoA and feruloyl-CoA were the only cinnamoyl-CoA derivatives accepted as starter substrates. PcPKS2 did not accept isobutyryl-CoA, isovaleryl-CoA or acetyl-CoA as substrate. DNA gel blot analysis indicated that there are two to four PcPKS2 copies in the P. cuspidatum genome. RNA gel blot analysis revealed that PcPKS2 is highly expressed in the rhizomes and in young leaves, but not in the roots of the plant. PcPKS2 transcripts in leaves were induced by pathogen infection, but not by wounding.

[Metabolic engineering of terpenoids in plants].

Terpenoids are present in all organisms but are especially abundant in plants, with more than 30,000 compounds. Not only do they play an important role in the life of plant, but also have high commercial values. However, the content of many important terpenoids in plant is very low. Therefore, how to improve the inefficient production of terpenoids is an urgent task. Metabolic engineering has been one of the most potential technologies to improve terpenoids production in recent years, following the study of metabolic pathway and regulation mechanism of terpenoids. Although there are some breakthroughs, metabolic engineering of terpenoids is still full of challenges because of the lack of knowledge on metabolic control of most terpenoids. Functional genomics approaches, including transcriptomics, proteomics and metabolomics, are potential tools for exploring of metabolic engineering. Integrating transcriptomics and metabolomics is an effective way to discover new genes involved in metabolic pathway. In this paper, the representative research outcomes about the metabolic engineering of terpenoids in plant were reviewed concisely and then the application of functional genomics approaches to study metabolic pathway and regulation mechanism of terpenoids and the strategies for metabolic engineering of terpenoids were discussed.

Molecular cloning and overexpression of a novel UDP-glucosyltransferase elevating salidroside levels in Rhodiola sachalinensis.

Salidroside is a novel effective adaptogenic drug extracted from the medicinal plant Rhodiola sachalinensis A. Bor. Because this plant is a rare resource and has low yield, there is great interest in enhancing the production of salidroside. In this study, a putative UDP-glucosyltransferase (UGT) cDNA, UGT73B6, was isolated from Rhodiola sachalinensis using a rapid amplification of cDNA ends (RACE) method. The cDNA was 1,598 bp in length encoding 480 deduced amino acid residues with a conserved UDP-glucose-binding domain (PSPG box). Southern blot analysis of genomic DNA indicated that UGT73B6 existed as a single copy gene in the R. sachalinensis genome. Northern blot analysis revealed that transcripts of UGT73B6 were present in roots, calli and stems, but not in leaves. The UGT73B6 under 35S promoter with double-enhancer sequences from CaMV-Omega and TMV-Omega fragments was transferred into R. sachalinensis via Agrobacterium tumefaciens. PCR, PCR-Southern and Southern blot analyses confirmed that the UGT73B6 gene had been integrated into the genome of transgenic calli and plants. Northern blot analysis revealed that the UGT73B6 gene had been expressed at the transcriptional level. High performance liquid chromatography (HPLC) analysis indicated that the overexpression of the UGT73B6 gene resulted in an evident increase of salidroside content. These data suggest that the cloned UGT73B6 can regulate the conversion of tyrosol aglycon to salidroside in R. sachalinensis. This is the first cloned glucosyltransferase gene involved in salidroside biosynthesis.

[Advances in sesquiterpene synthases cyclases of Artemisia annua].

Artemisinin,a new and a very potent antimalarial drug, is produced by the plant Artemisia annua L. with a very low yield ranging from 0.01% to 0.8% on a dry-weight basis. This makes artemisinin an expensive drug. Several studies reported chemical synthesis of the artemisinin, but none of them seems a viable economical alternative compared with the isolation of artemisinin from the plant. Hence, a higher artemisinin concentration in the plant is necessary for cheap antimalarial drug production. Many types of cyclic sesquiterpenes in Artemisia annua have been characterized to date, each derived from the common cyclic precursor FDP in a reaction catalyzed by a sesquiterpene synthase. Sesquiterpene synthases are widely regarded as the rate-determining regulatory enzymes in the pathways they participate, and a number of sesquiterpene synthases have been cloned from Artemisia annua up to now. This report is a brief review on the following sesquiterpene synthases: epi-cedrol synthase, amorpha-4,11-diene synthase, beta-caryophyllene synthase, (E)-beta-farnesene synthase, germacrene A synthase, as well as a new sesquiterpene synthase whose function remains largely unknown. The report is of help for a better understanding of metabolic engineering of Artemisia annua.

Metabolic engineering of terpenoids in plants / 生物工程学报

Terpenoids are present in all organisms but are especially abundant in plants, with more than 30,000 compounds. Not only do they play an important role in the life of plant, but also have high commercial values. However, the content of many important terpenoids in plant is very low. Therefore, how to improve the inefficient production of terpenoids is an urgent task. Metabolic engineering has been one of the most potential technologies to improve terpenoids production in recent years, following the study of metabolic pathway and regulation mechanism of terpenoids. Although there are some breakthroughs, metabolic engineering of terpenoids is still full of challenges because of the lack of knowledge on metabolic control of most terpenoids. Functional genomics approaches, including transcriptomics, proteomics and metabolomics, are potential tools for exploring of metabolic engineering. Integrating transcriptomics and metabolomics is an effective way to discover new genes involved in metabolic pathway. In this paper, the representative research outcomes about the metabolic engineering of terpenoids in plant were reviewed concisely and then the application of functional genomics approaches to study metabolic pathway and regulation mechanism of terpenoids and the strategies for metabolic engineering of terpenoids were discussed.

Advances in sesquiterpene synthases cyclases of Artemisia annua / 生物工程学报

Artemisinin,a new and a very potent antimalarial drug, is produced by the plant Artemisia annua L. with a very low yield ranging from 0.01% to 0.8% on a dry-weight basis. This makes artemisinin an expensive drug. Several studies reported chemical synthesis of the artemisinin, but none of them seems a viable economical alternative compared with the isolation of artemisinin from the plant. Hence, a higher artemisinin concentration in the plant is necessary for cheap antimalarial drug production. Many types of cyclic sesquiterpenes in Artemisia annua have been characterized to date, each derived from the common cyclic precursor FDP in a reaction catalyzed by a sesquiterpene synthase. Sesquiterpene synthases are widely regarded as the rate-determining regulatory enzymes in the pathways they participate, and a number of sesquiterpene synthases have been cloned from Artemisia annua up to now. This report is a brief review on the following sesquiterpene synthases: epi-cedrol synthase, amorpha-4,11-diene synthase, beta-caryophyllene synthase, (E)-beta-farnesene synthase, germacrene A synthase, as well as a new sesquiterpene synthase whose function remains largely unknown. The report is of help for a better understanding of metabolic engineering of Artemisia annua.

Studies on the effects of fpf1 gene on Artemisia annua flowering time and on the linkage between flowering and artemisinin biosynthesis.

The flowering promoting factor1 ( fpf1) from Arabidopsis thaliana was transferred into Artemisia annua L. via Agrobacterium tumefaciens. The fpf1 gene was firstly inserted in the binary vector pBI121 under the control of CaMV 35S promoter to construct the plant expression vector pBIfpf1, then leaf explants of A. annua were infected with A. tumefaciens LBA4404 containing pBIfpf1, and induced shoots. Transgenic plants were obtained through the selection with kanamycin. PCR, PCR-Southern and Southern blot analyses confirmed that the foreign fpf1 gene had been integrated into the A. annua genome. RT-PCR and RT-PCR-Southern analyses suggested that the foreign fpf1 gene had expressed at the transcriptional level. Under short-day conditions, the flowering time of fpf1 transgenic plants was about 20 days earlier than the non-transformed plants; however, no significant differences were detected in artemisinin content between the flowering transgenic plants and the non-flowering non-transgenic plants. These results showed that flowering is not a necessary factor for increasing the artemisinin content, furthermore, there may be no direct linkage between flowering and artemisinin biosynthesis.

[Advances in molecular regulation of artemisinin biosynthesis].

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.

Advances in molecular regulation of artemisinin biosynthesis / 生物工程学报

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.

Effects of ipt gene expression on the physiological and chemical characteristics of Artemisia annua L.

An isopentenyl transferase gene (ipt) from T-DNA was transferred into Artemisia annua L. via Agrobacterium tumefaciens. The ipt gene was placed in a binary vector under the control of the CaMV 35S promoter. Leaf explants were infected with A. tumefaciens LBA4404 containing pBIipt to induce the buds. Nineteen shoot lines were selected, which were resistant to kanamycin. Polymerase chain reactions and Southern blotting confirmed that at least five shoot lines contained the foreign gene. The results of RT-PCR and Northern blotting analyses suggested that the foreign ipt gene of the transgenic shoot was expressed. Cytokinins, chlorophyll and artemisinin contents were found increased at different degree. Content of cytokinins (iPA and iP) was elevated 2- to 3-fold, chlorophyll increased 20-60% and artemisinin increased 30-70% compared with the control plants, respectively. A direct correlation was found between the contents of cytokinins, chlorophyll and artemisinin. This may be the first report on the relationship between endogenous cytokinin content and the production of secondary metabolites in plants.