[关键词]
[摘要]
目的 分析研究黄花蒿3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)启动子的活性表达及其核心序列。方法 以β-葡萄糖苷酸酶(GUS)基因作为报告基因,通过构建包括HMGR启动子全长序列在内的5个不同长度表达载体,并采用农杆菌介导法转化烟草以及对转化烟草进行GUS染色。结果 转HMGR启动子烟草的根、茎、叶、花、果荚均发现蓝色;脱水和高温胁迫会降低该启动子的表达,低温胁迫对其影响不明显;启动子片段AaGPX1、AaGPX2和AaGPX3具有驱动GUS报告基因表达的功能,而AaGPX4和AaGPX5则未有此功能。结论 HMGR启动子在烟草整个生长期内均能够稳定表达;该启动子对低温不敏感,而对高温和脱水条件敏感;该启动子的核心序列在P-HMGR-3区域,而AaGPX2与AaGPX3片段的非重叠区则存在与HMGR启动子活性强弱相关的调控元件。
[Key word]
[Abstract]
Objective To analyze the expression character, screen the core sequence of 3-hydroxy-3-methyl glutaryl coenzyme A (HMGR) promoter from Artemisia annua, and provide a theoretical basis for the efficient biosynthesis of artemisinin. Methods In this study, β-glucoside acid enzyme (GUS) gene was used as the report gene and constructed five different length expression vectors, including the whole length of HMGR promoter, based on the cloned HMGR promoter sequences, and successfully obtained transgenic plants by the method of agrobacterium mediation transform tobacco, then the tobacco was stained by GUS. Results The GUS staining showed that the blue color could detect in the roots, stems, leaves, flowers, and fruit pods. And the leaves in the lower part could easily get the blue color than the leaves in the top part, this may due to that the accumulation of HMGR metabolite product is more and the permeability in leaves is better in the mature leaves. The results also showed that the color showed shallow in heat and drought stresses, and with no significant difference in cold stress, the tobacco leaves with paragraph of P-HMGR-1, P-HMGR-2, and P-HMGR-3 showed blue color but did not find blue in leaves with paragraph of P-HMGR-4, P-HMGR-5. Conclusion HMGR promoter has a stable expression during the whole growth period of tobacco. HMGR promoter was not sensitive with cold environment, but sensitive with heat and draught resistance. The key area of HMGR promoter was in P-HMGR-3. And there are some regulatory elements but no necessary part of HMGR promoter between the non overlap area of P-HMGR-3 and P-HMGR-2.
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[基金项目]
国际科技合作与交流专项“青蒿素低成本生产关键技术合作创新与转化”(2014DFA32160)