黄花蒿ADS启动子的分离及表达特性分析

Isolation and characterization of promoter of ADS from Artemisia annua

目的:从黄花蒿中分离鉴定青蒿素生物合成途径关键酶——紫穗槐二烯合酶编码基因(ADS)的启动子序列并研究其表达特性,藉此探索提高该基因表达量并进一步促进青蒿素合成的途径。方法:采用PCR法从黄花蒿DNA中分离ADS 5'端非翻译区(5'UTR)序列,构建与GUS报告基因融合的植物表达载体,通过农杆菌介导转化烟草。GUS组织化学染色法和分光光度法分别定性和定量检测ADS 5'UTR序列调控GUS基因在正常条件和胁迫条件下的表达。结果:从黄花蒿中分离出2 448 bp的ADS 5'UTR序列,获得与GUS基因融合表达的转基因烟草并检测到GUS活性。GUS活性定量检测结果显示,在4℃和紫外辐射条件下,转化烟草GUS活性分别提高1.6,2.2倍。结论:从黄花蒿分离出的ADS 5'UTR序列具有启动子功能并且可能具有环境诱导表达特性。

Objective： To 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 famesyl diphosphate, and accel- erate the artemisinin synthesis, the promoter of ADS was isolated and characterized. Method： 5＇ untranslated regions of ADS were iso- lated from Artemisia annua with PCR. For functional characterization, the isolated fragment was fused with GUS reporter gene and intro- duced into Nicotiana tabacum by Agrobacterium-mediated transformation. The GUS expression regulated by 5＇ untranslated regions of ADS in transgenie N. tabacum under the normal or stressed conditions were detected by histoehemical staining and quantitative spectro- photometry assay. Result： The 2 448 bp DNA fragment upstream of ADS coding sequence was isolated from A. annua and introduced into N. tabacum. Histoehemical staining showed that the isolated fragment conferred stable GUS expression in transgenie plants. The quantitative results showed that the GUS activity in transgenic tobacco plants treated by low-temperature （4 ℃ ） and ultraviolet irradia- tion were 1.6 and 2. 2 folds higher than that in the controls. Conclusion： It was suggested that the isolated fragment had promoter ac- tivity and maybe responsive to adverse environmental stresses.