A high-efficiency Agrobacterium-mediated transient expression system in the leaves of Artemisia annua L.
A high-efficiency Agrobacterium-mediated transient expression system in the leaves of Artemisia annua L.
复制标题
农杆菌介导的青蒿叶高效瞬时表达系统。
DOI：
10.1186/s13007-021-00807-5
复制
发表时间：
2021-10-16
期刊：
影响因子：
5.1
通讯作者：
Tang K
中科院分区：
文献类型：
--
作者：
Li Y;Chen T;Wang W;Liu H;Yan X;Wu-Zhang K;Qin W;Xie L;Zhang Y;Peng B;Yao X;Wang C;Kayani SI;Fu X;Li L;Tang K
农杆菌介导的瞬时转化技术因其简单、快速、高效等优点，在植物基因功能研究中得到了广泛应用。尽管人们在青蒿瞬时表达方面做了大量的工作，但建立一个快速、易操作的青蒿瞬时转化系统，实现高通量的基因功能鉴定仍是一个重要的研究方向。《年鉴》仍然具有挑战性。A. annua是农杆菌注射的理想候选物。EHA 105是建立瞬时表达系统的最佳菌株。0.005%浓度的Triton X-100的补充大大提高了瞬时表达频率。GUS组织化学染色结果显示，报告基因在转化过程中至少能维持一周的瞬时表达水平。双荧光素酶（Dual-LUC）瞬时检测结果表明，花椰菜花叶病毒35 S（CaMV 35 S）启动子及其衍生物在A. annua和烟草。以. annua中，CaMV 35 S启动子具有与双CaMV 35 S启动子相当的活性，而在烟草中，CaMV 35 S表现出约50%的双CaMV 35 S启动子活性。另外，尽管来自GoldenBraid Kit 2.0的CaMV 35 S启动子和双CaMV 35 S启动子在烟草中显示出高活性强度，但它们在瞬时表达的A. annua。同时对UBQ 10启动子和内源UBQb启动子的活性进行了研究。此外，利用我们的瞬时表达系统，证实了AaGSW 1和AaORA对AaCYP 71 AV 1启动子的反式激活。Dual-LUC分析表明，AaHD 8激活了两个腺毛特异性脂质转运蛋白基因AaLTP 1和AaLTP 2的表达，表明AaLTP 1和AaLTP 2可能是AaHD 8参与腺毛起始和角质层形成以及青蒿素分泌的下游基因。annua。建立了一种简单、快速、重复性好、高效、低成本的拟南芥瞬时转化体系。annua是开发的。该方法为基因亚细胞定位、启动子活性和转录激活等基因功能研究提供了新的途径。annua，避免了异源系统中基因表达导致的异常表型。在线版本包含补充材料，可通过10.1186/s13007-021-00807-5获得。
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. The online version contains supplementary material available at 10.1186/s13007-021-00807-5.
登录
查看更多内容
影响因子：
4.9
作者：
Adhikari, Prakash Babu;Han, Jung Yeon;Choi, Yong Eui
通讯作者：
Choi, Yong Eui
农杆菌介导的中间柠条瞬时表达系统的开发以及 CiDREB1C 在胁迫反应中的表征
影响因子：
5.3
作者：
Liu, Kun;Yang, Qi;Li, Guojing
通讯作者：
Li, Guojing
影响因子：
56.9
作者：
BENFEY, PN;CHUA, NH
通讯作者：
CHUA, NH
影响因子：
1.6
作者：
Aida, Ryutaro;Narumi, Takako;Shibata, Michio
通讯作者：
Shibata, Michio
影响因子：
7.2
作者：
Grefen, Christopher;Donald, Naomi;Blatt, Michael R.
通讯作者：
Blatt, Michael R.