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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 isolate and identify the promoter sequence of the key enzyme of artemisinin biosynthesis pathway (ADS) from Artemisia annua L. and to study its expression characteristics so as to explore and improve the expression level of this gene Ways to promote the synthesis of artemisinin. Methods: The 5 ’untranslated region (5’UTR) of ADS was isolated from A. annua DNA by PCR. The plant expression vector fused with GUS reporter gene was constructed and the tobacco was transformed by Agrobacterium tumefaciens. GUS histochemical staining and spectrophotometry were used to qualitatively and quantitatively detect the expression of GUS gene in ADS 5’UTR sequence under normal and stress conditions respectively. Results: The 2 448 bp ADS 5’UTR sequence was isolated from Artemisia annua and the transgenic tobacco fused with GUS gene was obtained and GUS activity was detected. The results of quantitative analysis of GUS activity showed that GUS activity of transformed tobacco increased by 1.6 and 2.2 times respectively under 4 ℃ and UV radiation. Conclusion: The ADS 5’UTR sequence isolated from Artemisia annua has promoter function and may have environmental-induced expression characteristics.