【摘要】 目的:研究低温处理对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含量;硫酸钛沉淀法和N,N-二甲基-对-亚硝基苯胺漂白测定法分别检测黄花蒿叶片过氧化氢(H2O2)和单线态氧(1O2)含量;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4h后黄花蒿叶片中1O2和H2O2含量升高,并伴随青蒿素含量和CAT活性逐步提高,4、24和48h后青蒿素含量分别提高20%、65%和80%;4℃处理24h后,青蒿素合成相关基因[3-羟基-3-甲基戊二酰辅酶A还原酶基因(HMGR)、法呢基焦磷酸合酶基因(FPS)、紫穗槐二烯合酶基因(ADS)、细胞色素P450单加氧酶基因(CYP71AV1)、细胞色素P450氧化还原酶基因(CPR)、青蒿醛Δ11(13)双键还原酶基因(DBR2)]的表达普遍上调,其中ADS的表达提高16倍;而青蒿素合成竞争途径的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。更多还原

【Abstract】 OBJECTIVE:To investigate the effects of low temperature on the biosynthesis pathway of artemisinin and other terpenoids.METHODS:After 4℃ treatment,the content of artemisinin was determined 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-dimethyl-nitrosoaniline.The catalase(CAT) activity was determined by ultraviolet spectrophotometry.The transcriptional levels of artemisinin biosynthetic and the expression of competitive enzymes encoding genes were quantitatively assayed by real time fluorescence quantitative-PCR.RESULTS:At 4 h after treatment,the increase of 1O2 and H2O2 was followed by the increase of CAT activity and artemisinin content.The contents of artemisinin were increased by 20%,65% and 80% at 4,24 and 48 h after treatment,respectively.The expression levels of artemisinin biosynthetic genes,such as HMGR,FPS,ADS,CYP71AV1,CPR and DBR2,were up-regulated after 4 ℃ treatment for 24 h.The increment of ADS mRNA level was 16 times higher than those under 25 ℃.Whereas the expression of(-caryophyllene synthase gene(CS) encoded the competitive enzyme dramatically decreased about 20-fold.CONCLUSION:These results indicate that low temperature induce artemisinin biosynthesis by increasing the conversion of the precursors into artemisinin caused by the increase 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.更多还原