农杆菌和根瘤菌--马铃薯介导的马铃薯转化与葡萄染色诱导素基因的启动子活性
Summary
在这里, 我们提出了两个协议, 以改造马铃薯植物。农杆菌的转化导致一个完整的转基因植物, 而农杆菌根状茎产生转基因毛状根在野生类型的芽, 可以自我繁殖。然后, 我们通过 GUS 染色检测转化根系中的启动子活性。
Abstract
农杆菌是获得转基因植物最广泛使用的方法之一, 因为它有能力将自己的 t-dna 转移并整合到植物的基因组中。在这里, 我们提出了两个转化系统, 基因改造马铃薯 (刺过木豆)植物。在肿瘤转化过程中, 叶子被感染, 转化后的细胞被选择, 一个新的完整的转化植物在18周内被再生。在根瘤菌转化过程中, 茎通过注射针头感染细菌, 用红色荧光标记检测新出现的转化的多毛根, 去除未转化的根。在5-6 内, 所产生的植物是一种野生类型的芽的组合, 具有完全发育的转化的毛状根。为了增加生物量, 转化后的毛状根系可以被切除和自我繁殖。我们应用这两种农杆菌介导的转化方法来获得根系表达 gus 记者基因驱动的亚素生物合成基因启动子。提供 GUS 染色程序, 并允许细胞定位的启动子诱导。在这两种方法中, 转化后的马铃薯根系在亚皮内皮和外真皮中都显示了 GUS 染色, 此外,在 a. 根瘤菌转化的根系中, 在侧根的出现中也检测到了 gus 活性。这些结果表明, a. 根瘤菌可以成为研究根系中表达的基因的快速替代工具。
Introduction
除了经济利益, 转基因植物的产生有其自身的相关性, 在研究中可以证明基因的最终功能, 并更好地了解植物的生理和发育。植物 DNA 插入最广泛使用的方法是农杆菌介导的转化。农杆菌通过其诱导肿瘤 (ti) 质粒的作用, 能够在许多植物物种的受感染组织中产生冠状凝块。质粒包含一个 t-dna 区域, 具有一组基因, 这些基因将被整合到植物基因组中, 并诱导组织去分化 1,2。转基因在 T-DNA 内交换这些基因, 使植物进行了特定的修饰, 避免了表型效应3。为了促进对 T-DNA 的转基因克隆, T-DNA 区域在一个称为二元质粒的独立质粒中被切除, 而 Ti 质粒 (允许 T-DNA 转移和插入机制的毒力基因) 的其余基因则被切除。放置在辅助质粒中。对于植物生物技术研究来说, 由a. tumefaciens进行改造有几个优点: 它不需要昂贵的设备, 能够产生稳定和短暂的植物转化, 并且将少量的基因副本集成到4号染色体.然而, 对于大多数植物, 但不是拟南芥,建立稳定的转化物需要植物再生从一个单一或几个细胞使用外源植物激素, 使这一过程费力和耗时。根瘤菌也能够改变植物基因组, 产生毛茸茸的根或不定根在感染部位由于表达的滚动(根位点) 基因编码在根诱导 (ri) 质粒 5.虽然研究较少, 但根瘤菌也被用于获得转基因根。在这种情况下, a. 根瘤菌在 ri 质粒中含有原始的 t-dna, 并在二元质粒中含有第二个携带转基因的 t-dna。当感染部位在茎或下胚轴时, 可以获得一种复合植物, 新的多毛转基因根从野生类型的芽中出现。另外, 毛状转化的根可以在有碳源输入的培养基中在体外自主生长。当根是靶器官时, 使用根质体而不是肿瘤产生转基因组织的方法正在变得越来越重要, 因为不需要植物再生, 因此它的速度更快, 成本更低。以往的研究已经证明了这种方法, 用于根特异性基因的表型表征 6,7,8,9。
根据联合国粮食及农业组织 (FAO) 的数据, 马铃薯(Solanum tuberom而d) 是世界上第四大最重要的作物, 因为块茎作为维生素的良好来源, 对人类消费具有营养意义和矿物。因此, 马铃薯已成为农业生物技术的焦点, 也被认为是遗传和发展研究的良好生物模式10、11.马铃薯转化通过对亚素和蜡生物合成所涉及的基因的表征, 对亚化组织的分子机制有显著的贡献, 12、13、14 ,15,16, 17, 亚素单体运输18和转录条例19。亚铁蛋白烯基转移酶基因Fht是这些具有特征的生物合成基因之一;其下调对表皮保护的影响很大, 这与马铃薯块茎14中的亚、酸和蜡的发酵酯率大幅下降有关。此外, 在拟南芥的根部和种子中, 其假定的正位词 (asft/rwp1) 的敲除也证明了其在亚、核素20、21中生成烷基发酵的作用。在马铃薯中, Fht转录记者行和 FHT 抗体分别表明启动子活性和蛋白质分别位于外皮、内皮、phellogn 衍生物和受伤组织15。
在这项工作中, 我们详细介绍了一个协议, 使用a. 根瘤菌生产转基因毛状根, 保存在野生类型的芽, 产生复合马铃薯植物, 或切除在体外自主生长。我们还提供了使用a. tumefaciens获得完整转基因马铃薯植物的协议。作为一个案例研究, 用相同的二元载体转化的a. 根瘤菌和a. tumefaciens 被用来获得根系与fht启动子驱动 gus 记者基因的表达。报告并比较结果。
Protocol
Representative Results
Discussion
在马铃薯中, 获得稳定完整转基因植物的最常见系统使用农杆菌菌株的转化, 这些菌株需要利用外源植物激素进行器官生成。尽管基于农杆菌的协议有可能整合非 t-dna 载体序列 25, 但这种方法仍然是最简单、成本更低的方法, 可以改造马铃薯植物。在过去的几年里, 人们对a. 根瘤菌介导的转化的兴趣引起了研究人员的关注, 因为它允许在比使用a. 肿瘤更短…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 innovo y Ciencia 部长 (AGL2009-13745, FPI 向 PB 提供的赠款)、经济和竞争事务部的赠款和 FEDER 的资助 (AGLUS-36725, AGL2015-675-C2-R) 和 Girona 大学 (向 SF 提供博士补助金, 并向 sf 提供博士赠款)单一/)。提交人感谢 Inge Broer 博士 (德国罗斯托克大学土地使用研究所) 和 Saloméprat 博士 (西班牙马德里国家生物技术中心) 提供了 a. Rhizogenes 和a. tumefaciens 菌株,并提供了 Marçal Soler 博士和 Anna Plasencia 博士在启动a.根瘤菌转化实验方面得到的帮助和支持 (Toulouse III Paul Sabatier 大学-CNRS, 植物研究实验室, Castanet tolosan,法国)。作者感谢 Sara Gómez (UdG、Girona 的 Biologia 省、Girona) 在开展实验室工作和照料植物方面提供的宝贵协助, 并感谢 Ferran Fontdecaba 和 Carla Sánchez 在进行一些实验时提供了协助。他们的最终学位项目。
Materials
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Acetone |
Panreac |
1.310.071.21 |
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Acetosyringone |
Acros |
115540050 |
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Aquarium pump |
Prodac |
MP350 |
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Autoclave |
Ragpa Strelimatic |
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Bacteriological agar |
Lab Conda |
1800 |
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BAP |
Duchefa |
B0904 |
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Beef extract |
Lab Conda |
1700 |
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Plant growing cabinet |
Nuaire |
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Carbenicillin |
Duchefa |
C0109 |
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Cefotaxime sodium |
Duchefa |
C0111 |
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DMSO |
Merck |
1029310161 |
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Ecotron infors |
HT |
29378 |
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Ethanol |
Merck |
1,009,831,011 |
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Falcon tube |
Control tecnica |
CFT011500 |
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Ferricyanate |
Sigma |
101001081 |
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Ferrocyanate |
Sigma |
100979088 |
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Flask (8.06 cm diameter and 11.3 cm height) and plastic lid for in vitro culture |
Apiglass |
ref16 |
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GA3 |
Sigma |
G7645 |
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Gamborg B5 media |
Duchefa |
G0210 |
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Gelrite |
Duchefa |
G1101 |
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Glucosa |
Sigma |
G5767 |
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Kanamycin |
Sigma |
K1377 |
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Leukopor tape |
BSN Leukopor |
BDF47467 |
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Lupe |
Wild-Heerbrugg |
M420 |
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Magnetic shaker |
Agimatic |
7000243 |
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MES hydrate |
Sigma |
M2933-25G |
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MgSO4 |
Panreac |
131404 |
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Microscope |
Olympus |
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Minufugue centrifugue 5415R |
Eppendorf |
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Murashige and Skoog media |
Duchefa |
M0254.0050 |
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Na2HPO4 |
Panreac |
131679 |
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NAA |
Duchefa |
N0903 |
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NaCl |
Panreac |
131659 |
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NaH2PO4 |
Sigma |
58282 |
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NightSea Stereo |
SFA Moonting Adapter |
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Parafilm |
Anorsa |
PRFL-001-001 |
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Peptone |
Lab Conda |
1616 |
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Petri dishes (90 x 14) |
Anorsa |
200200 |
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pHmetre |
Crison |
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Phytotron |
Inkoa |
RFTI-R5485 |
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Plant Agar |
Duchefa |
P1001 |
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Refrigeratot |
Liebherr Medline |
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Rifampicin |
Duchefa |
R0146 |
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Spectinomycin |
Sigma |
59007 |
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Spectrophotometer |
Shimadzu |
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Square plates (120 x 120) |
Deltalab |
200204 |
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Streptomycin |
Sigma |
S6501 |
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Sucrose |
Panreac |
131621 |
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Surgical blades |
Swann-Morton |
201 |
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Surgical needle |
NIPRO |
015/0204 |
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Triptone |
Lab Conda |
1612 |
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Triton |
Serva |
37240 |
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Unimax 1010 shaker |
Heidolph |
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Vacuum |
Dinko |
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x-GlcA (5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, sodium salt anhydrous) |
Biosynth |
B-7398 |
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Yeast extract |
Lab Conda |
1702.00 |
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Zeatin riboside |
Sigma |
1001042850 |
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