Li Yongpeng, Chen Tiantian, Wang Wei, Liu Hang, Yan Xin, Wu-Zhang Kuanyu, Qin Wei, Xie Lihui, Zhang Yaojie, Peng Bowen, Yao Xinghao, Wang Chen, Kayani Sadaf-Ilyas, Fu Xueqing, Li Ling, Tang Kexuan
Joint International Research Laboratory of Metabolic and Developmental Sciences, Key Laboratory of Urban Agriculture (South) Ministry of Agriculture, Plant Biotechnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China.
Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
Plant Methods. 2021 Oct 16;17(1):106. doi: 10.1186/s13007-021-00807-5.
The Agrobacterium-mediated transient transformation, which proved effective in diverse plant species, has been widely applied for high-throughput gene function studies due to its simplicity, rapidity, and high efficiency. Despite the efforts have made on Artemisia annua transient expression, achieving high-throughput gene functional characterization basing on a fast and easy-manipulated transient transformation system in A. annua remains challenging.
The first pair of true leaves of A. annua is an ideal candidate for Agrobacterium injection. EHA105 was the optimal strain that can be used for the development of the transient expression system. The supplementation of Triton X-100 at a concentration of 0.005% greatly improved the transient expression frequency. According to the histochemical β-Glucuronidase (GUS) staining assay, high transient expression level of the reporter gene (GUS) maintained at least a week. Dual-luciferase (Dual-LUC) transient assays showed that the activity of cauliflower mosaic virus 35S (CaMV35S) promoter and its derivates varied between A. annua and tobacco. In A. annua, the CaMV35S promoter had comparable activity with double CaMV35S promoter, while in tobacco, CaMV35S exhibited approximately 50% activity of double CaMV35S promoter. Otherwise, despite the CaMV35S promoter and double CaMV35S promoter from GoldenBraid Kit 2.0 displayed high activity strength in tobacco, they demonstrated a very low activity in transiently expressed A. annua. The activity of UBQ10 promoter and endogenous UBQb promoter was investigated as well. Additionally, using our transient expression system, the transactivation of AaGSW1 and AaORA on AaCYP71AV1 promoter was confirmed. Dual-LUC assays demonstrated that AaHD8 activated the expression of two glandular secreting trichomes-specific lipid transfer protein genes AaLTP1 and AaLTP2, indicating that AaLTP1 and AaLTP2 might serve as downstream components of AaHD8-involved glandular trichome initiation and cuticle formation, as well as artemisinin secretion in A. annua.
A simple, rapid, good-reproducibility, high-efficiency and low-cost transient transformation system in A. annua was developed. Our method offered a new way for gene functional characterization studies such as gene subcellular localization, promoter activity and transcription activation assays in A. annua, avoiding the aberrant phenotypes resulting from gene expression in a heterologous system.
农杆菌介导的瞬时转化在多种植物物种中已证明有效，因其简单、快速且高效，已被广泛应用于高通量基因功能研究。尽管在青蒿瞬时表达方面已做出努力，但基于一种快速且易于操作的青蒿瞬时转化系统实现高通量基因功能表征仍具有挑战性。
青蒿的第一对真叶是农杆菌注射的理想候选对象。EHA105是可用于开发瞬时表达系统的最佳菌株。添加浓度为0.005%的Triton X-100可大大提高瞬时表达频率。根据组织化学β-葡萄糖醛酸酶（GUS）染色分析，报告基因（GUS）的高瞬时表达水平至少维持一周。双荧光素酶（Dual-LUC）瞬时分析表明花椰菜花叶病毒35S（CaMV35S）启动子及其衍生物在青蒿和烟草中的活性有所不同。在青蒿中，CaMV35S启动子与双CaMV35S启动子具有相当的活性，而在烟草中，CaMV35S的活性约为双CaMV35S启动子的50%。此外，尽管来自GoldenBraid Kit 2.0的CaMV35S启动子和双CaMV35S启动子在烟草中显示出高活性强度，但它们在青蒿瞬时表达中表现出非常低的活性。还研究了UBQ10启动子和内源性UBQb启动子的活性。此外，使用我们的瞬时表达系统证实了AaGSW1和AaORA对AaCYP71AV1启动子的反式激活。双荧光素酶分析表明AaHD8激活了两个腺毛分泌特异性脂质转移蛋白基因AaLTP1和AaLTP2的表达，表明AaLTP1和AaLTP2可能作为参与青蒿腺毛起始、角质层形成以及青蒿素分泌的AaHD8的下游成分。
开发了一种简单、快速、可重复性好、高效且低成本的青蒿瞬时转化系统。我们的方法为青蒿基因功能表征研究提供了一种新途径，如基因亚细胞定位、启动子活性和转录激活分析，避免了在异源系统中基因表达导致的异常表型。
