Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.
The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.
Plant genetic engineering is conventionally used for transferring beneficial traits to plants. In recent years, this technology has been applied to convert plants into bio-factories for the production of pharmaceutically important and commercially valuable proteins, many of which cannot be chemically synthesized and are very costly to produce using animal or microbial systems1. By the introduction of new genes, the plant’s own metabolism can be manipulated for the production of various biopharmaceuticals like antibodies, metabolic enzymes, hormones, antigens or vaccine2.
Established gene transfer technologies for plants are the Agrobacterium-mediated delivery3, bombardment with DNA-coated microprojectiles (biolistics)4, and electroporation5 or polyethylene glycol6 treatment of protoplasts. Techniques requiring protoplasts are generally avoided because they are time-consuming, cumbersome and yield inconsistent results7. As a result, virtually all plant modification work at present utilizes either Agrobacterium or microprojectiles for gene transfer. The Agrobacterium method is more extensively used but not applicable to many economically important plant species. Meanwhile, microprojectile bombardment is more versatile due to a broad range of susceptible plants but requires specialized equipment and often causes severe tissue damage. Furthermore, these methods involve either a random (biolistics) or complex (Agrobacterium) delivery mechanism and have variable transformation rates8. Hence, a novel plant transformation technology that is simple yet effective, and applicable to different plant types is required.
Currently, plants are subjected to genetic modification primarily by the delivery of exogenous DNA encoding a desired trait, rather than by direct delivery of a target protein. The higher stability of DNA over proteins is a prime advantage; nevertheless, potential problems associated with DNA delivery include the random insertion of exogenous DNA into the plant genome and unintended transmission of antibiotic resistance genes to pathogenic bacteria via horizontal gene transfer9. For genome-editing purposes, the ability to edit plant genomes without introducing foreign DNA into cells may circumvent regulatory concerns related to genetically modified plants. Thus, an alternative DNA-free strategy for the modification of plants by direct delivery of protein will be able to cater to these needs.
Here we introduce a peptide-based system, originally developed for human gene therapy10-14, for the targeted delivery of exogenous genes or proteins in intact plants. Peptides are able to protect DNA from nuclease degradation and can mediate gene transfer across cell as well as organellar membranes15-17. They also have diverse and tunable properties besides being non-cytotoxic18-20. More importantly, with the use of peptides, genes can be precisely targeted to intracellular organelles such as the mitochondria21 or plastids (chloroplasts)22 for expression-a task not achievable by biolistic or Agrobacterium-mediated transformation. Depending on the cargo type, this new plant modification technology can be exploited to deliver proteins23 and either express (plasmid21,24 or double-stranded DNA25) or down-regulate (double-stranded RNA26) specific genes within the plant, throughout its cytosolic space24-26 or within a specific organellar compartment21. The designed carrier peptides consist of a cationic domain in the form of either polylysine (K8) or polylysine-co-histidine (KH)9 for binding and/or condensation of negatively-charged cargoes, which is conjugated to cell penetrating (BP100 peptide) or mitochondria transit (Cytcox peptide) sequences.
已经凭经验确定的协议中的关键步骤进行了讨论。通过注射器渗入,基于肽的制剂引入到通过气孔植物叶内的空域。以确保溶液的最大吸收,渗透过程应进行时的植物有利于气孔开口即条件下,提供足够的水,并在光周期下。至于转染复合物的制备,对涉及两个肽(线粒体靶向DNA递送)的组合制剂,用于添加各成分的顺序是至关重要的,不应该被反转。
有许多可行的修改的过程。对于植物细胞的浸润另一种选择是使用真空浸润,这是能够在复杂的溶液引入整个植株和/或部分组织,包括顶端分生组织。对于此替代过程,苗浸没在转染溶液,并基于由真空产生的压力,所述肽 – 货物复合物通过气孔和进植物细胞(吉住,T。,未发表的数据)被迫。
瞬时基因表达的基础上,使用动物细胞的研究中,已被证明能增加与更高的DNA浓度29-31。要注意的是肽的DNA的比例会影响复合物的生物物理性质(大小,表面电荷)是非常重要的( 图4),从而影响转染效率;因此,最佳的比率需要甚至具有增加的DNA浓度被维持。
之一的在使用肽作为基因/蛋白输送剂中的主要优点是,它的顺序是适合于调谐到满足所希望的功能。例如,所述载体肽的线粒体靶向域在该螺柱描述y可能与本地化这些细胞器叶绿体或过氧化物酶体靶向序列所取代。而叶绿体转化使用基因枪法32-34在几种植物是可能的,既不是农杆菌也不是生物射弹方法能够将基因导入线粒体或其它细胞器除了核( 表2)。
有用于基于肽的转染没有刚性宿主范围限制,不像农杆菌基于方法( 表2)。到目前为止,对于拟南芥 , 烟草本塞姆氏和杨树的转染制剂已被优化( 表1),但也可以适用于烟草的方法中, 番茄品种微汤姆,稻(基于预备实验),和其它的单 – 和双子叶植物。
迄今为止,有在转基因大小没有已知的限制时肽用于一小号转染载体。与此相反,已发现大量的DNA分子,以减少的土壤杆菌介导的方法35,36的转化效率,以及用于已报道37-39的约200kb的转基因的大小上限。使用基因枪,在另一方面,大的DNA片段可以制备或输送到植物中38中剪切。虽然没有上限被确定为生物射弹转化,到目前为止,已发现物理约束来限制可被转移到远小于150 kb的40 DNA的大小。使用相比于采用任何农杆菌或微粒轰击现有方法,以上所讨论的用于基因转移的基于肽的系统的主要优点,归纳在表2中 。
一些限制这种方法存在,因为它主张。首先,有针对性的pDNA的递送至特定的细胞器,如MITochondria,虽然效率较低已被证实可以通过DNA结合,细胞穿透和细胞器转运序列的简单组合。转基因表达可以被检测到,通过共聚焦显微术,仅在细胞内的线粒体的一个小口。因此,进一步的改进是必要的:(ⅰ)提高整个小区/细胞器膜更配合物的易位,以及(ii)提高的解离和从载体肽的pDNA转移到靶细胞器中表达。其次,使用该DNA递送系统中,外源报道基因的瞬时表达,成功在细胞的胞质和线粒体隔室来实现的。在植物核/细胞器基因组中导入的基因的稳定掺入和表达都尚未建立,但是,由于缺乏合适的选择策略。
虽然承认有改善或再经过D区才有发展,这里所描述的肽衍生战略仍然是一个简单而通用的技术已经铺平了交付各种货物到不同植物类型的方式。
The authors have nothing to disclose.
作者要感谢来自日本科学技术振兴机构的探索性研究的先进技术(JST-ERATO)中,新能源产业技术综合开发机构(NEDO)和跨部级战略创新促进计划(SIP),日本的资金。
(KH)9-BP100 peptide | Custom-synthesized by RIKEN Brain Science Institute | N/A | Sequence: KHKHKHKHKHKHKHKH- KHKKLFKKILKYL |
(BP100)2-K8 peptide | Custom-synthesized by RIKEN Brain Science Institute | N/A | Sequence: KKLFKKILKYLKKLFKKIL- KYLKKKKKKKK |
BP100 peptide | Custom-synthesized by RIKEN Brain Science Institute | N/A | Sequence: KKLFKKILKYL |
Cytcox-(KH)9 peptide | Custom-synthesized by RIKEN Brain Science Institute | N/A | Sequence: MLSLRQSIRFFKKHKHKH- KHKHKHKHKHKH |
P35S-GFP(S65T)-TNOS and P35S-RLuc-TNOS | N/A | N/A | Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of CaMV 35S constitutive promoter (Ref: Lakshmanan et al. Biomacromolecules 2013, 14, 10) |
pDONR-cox2:gfp and pDONR-cox2:rluc | N/A | N/A | Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of cox2 mitochondrial-specific promoter (Ref: Chuah et al. Sci Rep 2015, 5, 7751) |
dsDNA (PCR-amplified from pBI221-P35S-Rluc-TNOS) | N/A | N/A | Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of CaMV 35S constitutive promoter (Ref: Lakshmanan et al. Plant Biotechnol 2015, 32, 39) |
GFP5 siRNA | N/A | N/A | For RNA interference-mediated silencing of green fluorescent protein (Ref: Numata et al. Plant Biotechnol J 2014, 12, 1027) |
CHS siRNA | N/A | N/A | For RNA interference-mediated silencing of chalcone synthase (Ref: Numata et al. Plant Biotechnol J 2014, 12, 1027) |
1 mL and 10 mL Plastic Syringes | TERUMO Corporation | SS-01T, SS-10ESZ | |
1.5 mL Microcentrifuge Tube | AS ONE Corporation | 151212 | |
96-Well Flat-Bottom Plate | Asahi Glass Co., Ltd. | 3860-096 | |
Adhesive Tape | Sekisui Chemical Co., Ltd. | No. 835 | |
Alcohol Dehydrogenase | Sigma-Aldrich Co., LLC. | A-7011 | |
Atomic Force Microscope | Seiko Instruments Inc. | SPI3800, SPA 300HV | |
Atomic Force Microscope | Hitachi High-Tech Science Corporation | AFM5300E | |
BCA Protein Assay Kit | Thermo Fisher Scientific Inc. | 23227 | |
Cantilever | Hitachi High-Tech Science Corporation | K-A102001593 | |
Confocal Laser Scanning Microscope | Carl Zeiss | LSM700 | |
Cork Borer | Sigma-Aldrich Co., LLC. | Z165220 | For excision of leaves into 1-cm diameter disks |
Coverslip | Matsunami Glass Ind., Ltd. | C02261 | |
Cuvette | Sarstedt | 759116 | |
Folded Capillary Cell | Malvern Instruments, Ltd. | DTS1070 | |
Forceps | Shimizu Akira Inc. | Stainless pincet 150 | |
Homogenization Pestle | Ieda Trading Corp. | 9993 | |
Mica | Nisshin EM Co., Ltd. | LC23Z | |
Microcentrifuge | Beckman Coulter | BKA46472 | |
Microplate Reader | Molecular Devices Corporation | Spectra MAX M3 | |
Microscope Slide | Matsunami Glass Ind., Ltd. | S011120 | |
Pipette | Eppendorf Research® plus | 3120000909 | |
Pipette Tips | AS ONE Corporation | 2-5138-01, 2-5138-02, 2-5138-03 | |
Plastic Petri Dish | AS ONE Corporation | 1-7484-01 | |
Renilla Luciferase Assay Kit | Promega corporation | E2810 | |
Rhodamine B Isothiocyanate | Sigma-Aldrich Co., LLC. | 283924 | |
RNase-Free Water | Qiagen | 129112 | |
Sodium Carbonate | Wako Pure Chemical Industries, Ltd. | 199-01585 | |
Surgical Blade and Handle | FEATHER Safety Razor Co., Ltd. | Stainless steel No. 14 (blade), No. 3L (handle) | |
Syringe Tip Cap | Musashi Engineering Inc. | NC-3E | |
Weighing Balance | Sartorius | CPA225D | |
Zeta Potentiometer | Malvern Instruments, Ltd. | Zetasizer Nano-ZS |