诱导毛茸茸的根由农业细菌根- 在塔特里巴克麦的中介转化 （法戈皮鲁姆塔塔里库姆）
1Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences, 2Artemisinin Research Center,China Academy of Chinese Medical Sciences, 3College of Life Science,Huaibei Normal University, 4Economic Crop Research Institute Sichuan Academy of Agriculture Sciences, 5Research Center of Buckwheat Industry Technology,Guizhou Normal University
Summary
我们描述了一种通过农业细菌根诱导毛茸茸的根的方法 – 在塔特里麦（法戈皮鲁姆塔塔里库姆）中介导的转化。这可用于研究塔塔麦中继发代谢物的基因功能和产生，用于任何基因转化，或用于改良后的其他药用植物。
Abstract
塔塔麦 （TB） –Fagopyrum 榻地（L.） Gaertn] 拥有各种生物和药理活性，因为它含有丰富的二次代谢物，如黄酮类化合物，特别是鲁氏素。全球逐渐利用植物原体诱导药用植物中的毛根，以研究基因功能，提高继发代谢物的产量。在这项研究中，我们描述了一种在结核病中产生由根介导的毛根的详细方法。在7-10天时选择科蒂莱顿和低血节轴作为外植体，并感染携带二进制载体的A.根茎，这诱发了1周后出现的令人毛骨悚然的毛根。根据形态、抗性选择（卡那霉素）和记者基因表达（绿色荧光蛋白）确定产生的毛根转化。随后，转换后的毛根根据需要自我传播。同时，骨髓性细胞病（MYB）转录因子FtMYB116，利用A.根细胞介导的毛根转化为结核病基因组，以验证FtMYB116在合成黄酮类化合物中的作用。结果表明，FtMYB116显著推广了黄酮类相关基因的表达和黄酮类化合物（鲁汀和奎西汀）的产生率（p < 0.01），表明A.rhizo基因介导的毛根可以作为研究基因功能和次生代谢物产生的有效替代工具。本研究中描述的生成毛根的详细分步协议可在调整后用于任何基因转化或其他药用植物。
Introduction
塔特里麦 （TB） （法戈皮鲁姆塔塔里库姆（L.） 盖尔滕） 是属于法戈皮鲁姆属和家族 Polygonaceae1的二恶二甲。结核病作为一种中药同源性食品，由于其独特的化学成分和多种生物活动，对疾病产生了浓厚的兴趣。结核病主要富含碳水化合物、蛋白质、维生素和类胡萝卜素，以及多酚，如酚酸和黄酮类化合物1。黄酮类化合物的各种生物和药理活性，包括抗氧化、抗高血压2、抗炎以及抗癌和抗糖尿病特性，已被证明有3种。
植物杆菌是一种土壤细菌，通过感染伤口部位44、5，5有助于几种高等植物，特别是二叶草病的发展。这个过程是由诱导（Ri）质粒55、66中的T-DNA转移开始的，通常伴随着来自Ri质粒的外源基因的整合和表达以及产生毛根表型7的后续步骤。A. 根茎 –介导的转基因毛根，作为植物生物技术领域的一种强大工具，由于其稳定和高生产率和容易在短时间内获得，得到了最广泛的应用。此外，由A.根茎诱导的毛根有效地区分其拉皮性根发育和高分枝生长在无激素介质8。它们可用于多个研究领域，包括人工种子生产、根结核研究，以及研究与其他生物（如霉菌、线虫和根病原体77、9）9的相互作用。此外，毛茸茸根转化培养物已被广泛用作研究生化途径和化学信号的实验系统，并生产用作药品、化妆品和食品添加剂的植物二次代谢物8,8，10。在野生型毛茸茸的根部合成的有价值的二次代谢物，包括多叶生物碱、异丙酮、龙烷生物碱、二烯醇和黄酮类化合物，在众多物种中已经研究了几十年，如Panax人参11中的人参、阿美马伊1212中的科马林和TB2中的酚类化合物。
毛根已经产生使用A.根基因在79种植物从27个家族14。例如，在大豆15，16，16萨尔维亚17，普伦巴戈印度18，莲花雅波尼库斯19，和菊花（Cichorium intybus L.）中报道了A.rhizo基因介导的毛根转化。1520.结核病毛根转化也已调查2.关于由A.根基因介导的毛状根的开发，很少详细的协议，要么携带二进制向量。例如，Sandra等人21日介绍了一种在野生型芽中维持的转基因马铃薯毛根生产方法。完全发育的毛根可以在将A.rhizo基因注射到马铃薯植物的茎间5-6周后可视化。另一项研究也报道了由A.根茎诱导的转基因毛根系统，该基因含有黄麻中的gusA报告基因（科丘齐斯帽状体L.）。22.此外，Supaart等人23利用表达载体pBI121转化的转基因烟草毛根获得，该基因携带β1-四氢大麻素酸（THCA）合成酶的基因，以产生THCA。
然而，有效生成毛茸茸的根部转化的分步过程，特别是在结核病中，其表现相对较少。在这项研究中，我们描述了一个详细的协议，使用携带报告基因（GFP），选择性标记（Kan）和感兴趣的基因（b4，从我们组识别，但未发表的基因从基本螺旋环螺旋（bHLH）家族）产生毛茸茸的根基因转化在结核病。实验持续了5-6周，从种子的接种到毛茸茸的根的生成，涉及外植制剂、感染、凝育、亚培养和随后的传播。此外，A. 根茎基因包含携带骨髓性细胞变核转录因子116（FtMYB116）的二FtMYB116元质粒，用于确定FtMYB116能否促进在结核病基因和代谢水平上通过TB毛根转化积累类黄酮，尤其是鲁氏素。FtMYB116是一种光诱导转录因子，调节不同光照条件下鲁因的合成。黄酮合成酶（CHS），黄酮-3-羟基酶 （F3H），黄酮-3′-羟基酶 （F3‘H）和黄酮醇合成酶 （FLS）24是参与鲁金生物合成代谢途径的关键酶.因此，本研究证明了FtMYB116在TB毛根的过度表达，关键酶基因的表达，以及鲁汀和其他黄酮类化合物（如奎西汀）的含量。
Protocol
本研究中使用的结核病被命名为BT18，它起源于山西省农科院小杂粮研究中心培育的”金乔二号”品种。该协议的主要步骤如图1所示。 注：快速操作与植物相关的操作，并尽可能关闭培养皿，以避免枯萎和污染。除非另有说明，所有外植株孵育是在14小时光和10小时暗光期25°C下进行的。除非另有说明，否则所有与植物或细菌有关的操作都是在层压流量罩无?…
Representative Results
农业细菌根- 介导的结核毛根转化这项研究描述了为利用A.根茎获得基因转化的毛根而建立的分步协议。从结核病种子的接种到已鉴定的毛根的收获，大约需要5-6周的时间，图1（A-H）描述了一些关键步骤。A-H简单地说，消毒的壳种子接种（图1B），以实现更快的无菌发芽。A. 根茎（<strong class=…
Discussion
结核病已用于与遗传和代谢5,水平1，2，5，27，282,的继发代谢物有关的几项研究。,27128毛茸茸的根培养作为代谢物产生的独特来源，在代谢工程29中起着关键作用，并可用于通过插入相关基因来改变代谢途径。Kim等人2最初介绍建立结核毛根…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中央公益性研究机构ZXKT17002基础研究基金的支持。
Materials
| 2*Taq PCR MasterMix | Aidlab, China | PC0901 | |
| Agar powder | Solarbio Life Science, Beijing, China | A8190 | |
| Applied Biosystems 2720 thermo cycler | ThermoFisher Scientific, US | A37834 | |
| AS | Solarbio Life Science, Beijing, China | A8110 | Diluted in DMSO, 100 mM |
| binary vectors | ThermoFisher Scientific (invitrogen), US | / | pK7WG2D/pK7GWIWG2D (II) |
| Cefotaxime,sodium | Solarbio Life Science, Beijing, China | C8240 | Diluted in Water, 200 mg/mL |
| CF15RXII high-speed micro | Hitachi, Japan | No. 90560201 | |
| Diposable Petri-dish | Guanghua medical instrument factory, Yangzhou, China | / | |
| DYY-6C electrophoresis apparatus | Bjliuyi, Beijing China | ECS002301 | |
| EASYspin Plus Plant RNA Kit | Aidlab, China | RN38 | |
| ELGA purelab untra bioscience | ELGA LabWater, UK | 82665JK1819 | |
| Epoch Microplate Spectrophotometer | biotek, US | / | |
| Gateway BP/LR reaction enzyme | ThermoFisher Scientific (invitrogen), US | 11789100/11791110 | |
| HYG-C multiple-function shaker | Suzhou Peiying Experimental Equipment Co., Ltd. China | / | |
| Kan | Solarbio Life Science, Beijing, China | K8020 | Diluted in Water, 100 mg/mL |
| MLS-3750 Autoclave sterilizer | Sanyo, Japan | / | |
| MS salts with vitamins | Solarbio Life Science, Beijing, China | M8521 | |
| NaCl | Solarbio Life Science, Beijing, China | S8210 | |
| Other chemicals unstated | Beijing Chemical Works, China | ethanol, mercury bichloride, etc. | |
| PHS-3C pH meter | Shanghai INESA Scientific Instrument Co., Ltd, China | a008 | |
| Plant Genomic DNA Kit | TIANGEN BIOTECH (BEIJING) CO., LTD | DP305 | |
| Rifampin | Solarbio Life Science, Beijing, China | R8010 | Diluted in DMSO, 50 mg/mL |
| Spectinomycin | Solarbio Life Science, Beijing, China | S8040 | Diluted in Water, 100 mg/mL |
| Sucrose | Solarbio Life Science, Beijing, China | S8270 | |
| Trans2K DNA Marker | TransGen Biotech, Beijing, China | BM101-01 | |
| Tryptone | Solarbio Life Science, Beijing, China | LP0042 | |
| Whatman diameter 9 cm Filter paper | Hangzhou wohua Filter Paper Co., Ltd | / | |
| Yeast Extract powder | Solarbio Life Science, Beijing, China | LP0021 |
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Citer Cet Article
Mi, Y., Zhu, Z., Qian, G., Li, Y., Meng, X., Xue, J., Chen, Q., Sun, W., Shi, Y. Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat (Fagopyrum tataricum). J. Vis. Exp. (157), e60828, doi:10.3791/60828 (2020).