黄花蒿cyp71av1启动子的分离及表达特性分析

Isolation and expression characteristic of cyp71av1 promoter 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——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 Trying to find the ways to enhance the expression of cyp71av1 gene encoding cytochrome P450 mono-oxygenase which is a key enzyme in artemisinin biosynthesis pathway accelerating the artemisinin synthesis,the promoter of cyp71av1 was isolated and characterized.Methods 5’ untranslated regions of cyp71av1 were isolated from Artemisia annua with thermal asymmetric interlaced PCR.For functional characterization,the isolated fragments were fused with β-glucuronidase GUS reporter gene and introduced into Nicotiana tabacum by Agrobacterium-mediated transformation.The GUS expression regulated by 5’ untranslated regions of cyp71av1 in transgenic N.tabacum under the normal or stressed conditions were detected by histochemical staining and quantitative spectrophotometry assay.Results Two DNA fragments upstream of cyp71av1 coding sequence,a long fragment and a truncated fragment,were isolated from A.annua and introduced into N.tabacum respectively.Histochemical staining showed that two isolated fragments confered stable GUS expression in transgenic plants,and no significant difference was found between the two fragments on the GUS activity.The quantitative results also showed that the GUS activity in transgenic tobacco plants treated by dehydration,low-temperature（4 ℃）,and ultraviolet irradiation were 1.4 to 2.7 folds higher than that in the controls.Conclusion It suggests that the isolated fragments has promoter activity and may be responsive to adverse environmental stresses.