Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), Chongqing Engineering Research Centre for Sweet Potato, SWU-TAAHC Medicinal Plant Joint R&D Centre, School of Life Sciences, Southwest University, Chongqing, China.
Tobacco Breeding and Biotechnology Research Center, Yunnan Academy of Tobacco Agricultural Sciences, Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Kunming, China.
J Exp Bot. 2019 Sep 24;70(18):4835-4848. doi: 10.1093/jxb/erz220.
Basic helix-loop-helix (bHLH) proteins are the second largest family of transcription factors (TFs) involved in developmental and physiological processes in plants. In this study, 205 putative bHLH TF genes were identified in the genome of Artemisia annua and expression of 122 of these was determined from transcriptomes used to construct the genetic map of A. annua. Analysis of gene expression association allowed division of the 122 bHLH TFs into five groups. Group V, containing 15 members, was tightly associated with artemisinin biosynthesis genes. Phylogenetic analysis indicated that two bHLH TFs, AabHLH106 and AabHLH112, were clustered with Arabidopsis ICE proteins. AabHLH112 was induced by low temperature, while AabHLH106 was not. We therefore chose AabHLH112 for further examination. AabHLH112 was highly expressed in glandular secretory trichomes, flower buds, and leaves. Dual-luciferase assays demonstrated that AabHLH112 enhanced the promoter activity of artemisinin biosynthesis genes and AaERF1, an AP2/ERF TF that directly and positively regulates artemisinin biosynthesis genes. Yeast one-hybrid assays indicated that AabHLH112 could bind to the AaERF1 promoter, but not to the promoters of artemisinin biosynthesis genes. Overexpression of AabHLH112 significantly up-regulated the expression levels of AaERF1 and artemisinin biosynthesis genes and consequently promoted artemisinin production.
基本螺旋-环-螺旋 (bHLH) 蛋白是参与植物发育和生理过程的第二大转录因子 (TF) 家族。在这项研究中，在黄花蒿基因组中鉴定了 205 个推定的 bHLH TF 基因，并从用于构建黄花蒿遗传图谱的转录组中确定了其中 122 个的表达。基因表达关联分析将 122 个 bHLH TF 分为五组。第 V 组包含 15 个成员，与青蒿素生物合成基因紧密相关。系统发育分析表明，两个 bHLH TF，AabHLH106 和 AabHLH112，与拟南芥 ICE 蛋白聚类。AabHLH112 受低温诱导，而 AabHLH106 不受低温诱导。因此，我们选择 AabHLH112 进行进一步研究。AabHLH112 在腺毛分泌毛状体、花蕾和叶片中高度表达。双荧光素酶测定表明，AabHLH112 增强了青蒿素生物合成基因和 AP2/ERF TF AaERF1 的启动子活性，后者直接正向调控青蒿素生物合成基因。酵母单杂交测定表明，AabHLH112 可以结合到 AaERF1 启动子上，但不能结合到青蒿素生物合成基因的启动子上。AabHLH112 的过表达显著上调了 AaERF1 和青蒿素生物合成基因的表达水平，从而促进了青蒿素的产生。
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