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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——细胞色素P450单加氧酶编码基因(cyp71av1)的启动子序列并研究其表达特性,探索提高该基因表达量并进一步促进青蒿素合成的途径。方法采用热不对称嵌套PCR法从黄花蒿DNA中分离cyp71av1 5’端非翻译区序列,构建与β-葡萄糖苷酸酶(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。采用GUS组织化学染色法和分光光度法分别定性和定量检测cyp71av1 5’端非翻译区序列调控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长短2个cyp71av1 5’端非翻译区序列,分别获得与GUS基因融合表达的转基因烟草,均能检测到GUS活性且两者无明显差异。GUS活性定量检测结果还显示,在脱水、4℃和紫外辐射条件下,转化烟草GUS活性提高1.4～2.7倍。结论从黄花蒿分离出的2个cyp71av1 5’端非翻译区序列都具有启动子功能,并且具有环境诱导表达特性。
OBJECTIVE To isolate and identify the promoter of cyp71av1, a key enzyme in artemisinin biosynthesis pathway, from Artemisia annua, and to explore the expression characteristics of cyp71av1 gene, explore the expression of this gene and further promote Artemisia annua Synthesis of the way. Methods Thermo-asymmetric nested PCR was used to separate the 5 ’untranslated region of cyp71av1 from A. annua DNA and construct a plant expression vector fused with β-glucuronidase (GUS) reporter gene. Agrobacterium-mediated transformation of tobacco . GUS histochemical staining and spectrophotometry were used to qualitatively and quantitatively detect the expression of GUS gene regulated by 5 ’untranslated region of cyp71av1 under normal and stress conditions respectively. Results The sequence of the 5 ’untranslated region of cyp71av1 was isolated from Artemisia annua, and the GUS activity was detected in transgenic tobacco with GUS gene fusion respectively. There was no significant difference between them. GUS activity quantitative test results also showed that under dehydration, 4 ℃ and UV radiation conditions, transformed tobacco GUS activity increased by 1.4 to 2.7 times. Conclusion The two cyp71av1 5’-untranslated region sequences isolated from Artemisia annua L. all have promoter function and have environmental-induced expression characteristics.