摘要
目的研究低温处理对黄花蒿中青蒿素及其他萜类合成通路的影响。方法以4℃为胁迫条件,利用高效液相色谱法测定青蒿素量;硫酸钛沉淀法和N,N-二甲基-对-亚硝基苯胺漂白反应分别检测黄花蒿叶片过氧化氢(H2O2)和单线态氧(1O2)量;紫外分光光度法检测过氧化氢酶(CAT)活性;实时荧光定量PCR技术定量分析青蒿素合成途径及竞争途径关键酶基因的表达。结果 4℃处理4 h后黄花蒿叶片中1O2和H2O2量升高,并伴随着青蒿素量和CAT活性逐步提高,4、24、48 h后青蒿素量分别提高20%、65%、80%;4℃处理24 h后,青蒿素合成相关基因(HMGR、FPS、ADS、CYP71AV1、CPR和DBR2)的表达普遍上调,其中ADS基因的表达提高16倍;而青蒿素合成竞争途径酶(β-石竹烯合酶)基因(CS)表达则下调近20倍。结论低温刺激可能通过产生高浓度活性氧(ROS)促进青蒿素合成前体转化,上调青蒿素合成相关基因表达并抑制竞争途径基因表达等途径促进青蒿素合成。
Objective To investigate the effects of low temperature on the biosynthesis of artemisinin and other terpenoids in Artemisia annua. Methods After 4 ℃ treatment, A. annua leaves were collected for artemisinin analysis by HPLC. Hydrogen peroxide （H2O2） and singlet oxygen （^1O2） production were detected by Ti（SO4）2 precipitation and the means of bleaching of N, N-dimethylnitrosoaniline. The catalase （CAT） activity was determined by ultraviolet spectrophotometry. The gene expressions of key enzymes in artemisinin biosynthetic and competitive pathways were quantitatively assayed by real time fluorescence quantitative polymerase chain reaction （PCR）. Results At 4 ~C after treatment for 4 h, the burst of ^1O2 and H202 was followed by the increase of CAT activity and artemisinin content. The concentrations of artemisinin were 20%, 65%, and 80% higher than those of the control group after treatment for 4, 24, and 48 h, respectively. The expression levels of artemisinin biosynthetic genes, comprising HMGR, FPS, ADS, CYPT1AV1, CPR, and DBR2, were up-regulated compared with the control group at 4 ℃ after treatment for 24 h. The increment of ADS mRNA level was 16 times higher than those under 25℃. Whereas the expression of β-caryophyllene synthase （CS） gene encoded the competitive enzyme utilizing the common precursor dramatically decreased about 20-fold. Conclusion These results indicate that low temperature could induce artemisinin biosynthesis by increasing the conversion of the precursors into artemisinin caused by the burst of reactive oxygen species （ROS）, by the up-regulating the expression of genes involved in artemisinin biosynthesis, and by down-regulating the expression of gene involved in competitive pathway.
出处
《中草药》
CAS
CSCD
北大核心
2012年第2期350-354,共5页
Chinese Traditional and Herbal Drugs
基金
广东省自然科学基金资助项目(9145624536-4000003)
关键词
黄花蒿
青蒿素
低温诱导
活性氧
基因表达
Artemisia annua L.
artemisinin
low temperature-induced
reactive oxygen species （ROS）
gene expression
