大豆毛根转化基因功能分析
Summary
在这里，我们提出了一种高效生产转基因大豆毛根的方案。
Abstract
大豆（Glycine max）是一种有价值的农业作物，具有数千种工业用途。大豆根系是与土壤传播微生物相互作用的主要场所，这些微生物形成共生以固定氮和病原体，这使得涉及大豆根遗传学的研究对于提高其农业生产至关重要。大豆毛根（HRs）的遗传转化由根 茎农杆菌 菌株NCPPB2659（K599）介导，是研究大豆根系基因功能的有效工具，从开始到结束仅需2个月。在这里，我们提供了一个详细的协议，概述了在大豆HR中过表达和沉默目的基因的方法。该方法包括大豆种子灭菌，用K599感染子叶，以及选择和收获基因转化的HR进行RNA分离，并在必要时进行代谢物分析。该方法的通量足以同时研究多个基因或网络，并且可以在致力于长期稳定的转化方法之前确定最佳工程策略。
Introduction
大豆（Glycine max）是农业中最有价值的作物之一。它具有数以千计的商业和工业用途，例如食品，动物饲料，油，并作为制造原料的来源1。它与固氮土壤微生物（即根瘤菌）形成共生关系的能力进一步提升了研究大豆遗传学的重要性2。例如，微调大豆根系的固氮特性可以减少碳排放，并大大降低对氮肥的需求3。因此，特别是了解控制大豆根生物学方面的遗传学在农业和工业中具有广泛的应用。考虑到这些好处，有一个可靠的方案来分析大豆基因的功能是很重要的。
根癌农杆菌可能是植物遗传转化最常用的工具，因为它具有将转移DNA（T-DNA）整合到许多植物物种的核基因组中的能力。当农杆菌感染植物时，它将肿瘤诱导（Ti）质粒转移到宿主染色体中，导致在感染部位形成肿瘤。几十年来，农杆菌介导的转化已被广泛用于基因功能分析和修改作物性状4。尽管任何目的基因都可以通过根癌曲霉介导的转化轻松转移到宿主植物细胞中，但这种方法有几个缺点;它耗时、昂贵，并且需要对大豆等许多植物物种提供广泛的专业知识。虽然少数品种的大豆可以通过使用根癌农杆菌的子叶节点方法进行转化，但这种方法的低效率需要一种快速高效的替代遗传转化技术4,5。即使是非专家也可以使用这种根茎农杆菌介导的毛根（HR）转化方法来克服这些缺点。
HR转化是一种相对快速的工具，不仅用于分析基因功能，还用于生物技术应用，例如生产专门的代谢物和精细化学品，以及复杂的生物活性糖蛋白6。大豆HR的生产不需要广泛的专业知识，因为它们可以通过伤害子叶表面，然后接种根 茎农杆菌7来产生。 A. rhizogenes 表达由其Ti质粒编码的毒力（Vir）基因，该质粒转移，携带并将其T-DNA片段整合到植物细胞的基因组中，同时刺激异位根生长8。
与其他大豆基因表达系统（例如基于生物利斯特或根癌农杆菌的组织、细胞和器官培养转化）相比，HR表达系统具有几个优点。首先，HR在遗传上是稳定的，并且在无激素培养基上快速产生1,9,10。此外，HR可以产生相当于或大于天然根11,12的特化代谢物。这些优点使HRs成为与根癌曲霉不相容或需要特殊组织培养条件以形成相容组织的植物物种的理想生物技术工具。HR 方法是一种使用 RNA 测序13,14,15 分析蛋白质-蛋白质相互作用、蛋白质亚细胞定位、重组蛋白质生产、植物修复、诱变和全基因组效应的有效方法。它还可用于研究在工业中具有价值的专用代谢物的生产，包括甘氨酸，其药物可预防大豆对重要的微生物病原体疫霉，并在人类中具有令人印象深刻的抗癌和神经保护活性16,17。
本报告展示了一种简单、高效的生产大豆 HR 的方案。 与以前的 HR 转化方法相比，该方案通过在接种大豆子叶之前预先筛选根 茎变形 体中存在 Ti 质粒的 HR 形成率，显着提高了 （33%-50%）。我们通过转换几种过表达或沉默大豆转录因子基因的二元载体来证明该协议的适用性。
Protocol
Representative Results
Discussion
在过去的十年中，大豆HR方法已被开发为研究参与固氮22,23，生物和非生物胁迫耐受性24,25以及代谢物生物合成途径26,27的基因的有力工具。植物如何产生代谢物的知识对农业生产和制药工业有很多好处，因为它可用于研究参与介导对病原体的生化防御的基因网络<sup …
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究由加拿大自然科学和工程研究委员会（NSERC）资助号RGPIN-2020-06111和Brad Lace的慷慨捐赠。我们要感谢Wayne Parrott （佐治亚大学）提供K599农杆菌和初步方案，感谢Nakagawa&Hachiya实验室（岛根大学）提供pGWB2，pGWB6和pANDA35HK空载体。
Materials
| Acetosyringone | Cayman | 23224 | |
| Bleach | lavo | 21124 | |
| DMSO | Fisher bioreagents | 195679 | |
| Gelzan | Phytotech | HYY3251089A | |
| Hygromycin | Phytotech | HHA0397050B | |
| Isopropyl alcohol | Fisher chemical | 206462 | |
| Kanamycin | Phytotech | SQS0378007G | |
| LB powder | Fisher bioreagents | 200318 | |
| MS powder | Caisson labs | 2210001 | |
| Na2HPO4 | Fisher bioreagents | 194171 | |
| NaCl | Fisher chemical | 192946 | |
| Petri dishes | Fisherbrand | 08-757-11 | 100 mm x 25 mm |
| Phosphinothricin | Cedarlane | P034-250MG | |
| REDExtract-N-Amp PCR Kit | Sigma | R4775 | |
| Sucrose | Bioshop | 2D76475 | |
| Timentin | Caisson labs | 12222002 | |
| Vitamins | Caisson labs | 2211010 |
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