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
目的:建立 RP-HPLC 法测定通光藤药材中11α-O-顺芷酰基-12β-O-乙酰基-通光藤苷元 B 的含量。方法:采用酸水解法降解通光藤 C_(21)甾体苷,以反相高效液相色谱法测定11α-O-顺芷酰基-12β-O-乙酰基-通光藤苷元 B 含量,色谱柱为 Diamonsi...目的:建立 RP-HPLC 法测定通光藤药材中11α-O-顺芷酰基-12β-O-乙酰基-通光藤苷元 B 的含量。方法:采用酸水解法降解通光藤 C_(21)甾体苷,以反相高效液相色谱法测定11α-O-顺芷酰基-12β-O-乙酰基-通光藤苷元 B 含量,色谱柱为 Diamonsil ODS(250 mm×4.6 mm,5 μm);柱温为16℃;流动相为乙腈-水(45:55);流速为0.8 mL·min^(-1);检测波长为220 nm。结果:11α-O-顺芷酰基-12β-O-乙酰基-通光藤苷元 B 进样量在1.016～5.080μg范围内与峰面积积分值呈良好的线性关系,r=0.9999(n=5);平均回收率(n=6)为98.8%,RSD=1.9%。结论:方法简便、快速、准确,为通光藤药材的质量评价和新药开发提供科学依据。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.
基金supported by the Educational Science Foundation of Beijing (No. KM200510025009, KM200710025004)theNational Basic Research Development Program of China (No. 2006CB503807)
