TRTech-PGR: Agrobacterium-mediated transformation of the plastid genome

利用对大观霉素（一种用于叶绿体转化的选择性剂）高度敏感的植物，在拟南芥中实现了高频质体转化。目前拟南芥质体转化的瓶颈是难以从质体组织培养细胞中获得可育植株。利用农杆菌在卵母细胞中直接转化质体，并通过在选择性培养基上萌发种子来鉴定转基因事件，可以克服这种组织培养的局限性。为了实现这一目标，农杆菌将被重新设计，使T-DNA能够传递到母体生殖系细胞的质体中。该方法将避免组织培养和组织培养诱导的遗传变异，从而大大简化了获得转质体植物的过程。回避组织培养过程消除了在拟南芥中实践质体转化的专业知识的需要。因此，本文提出的研究将导致拟南芥质体基因组工程的广泛应用，结合现有的广泛基因组资源，将对生物技术的基础科学和应用产生重大影响。新的质体转化方案将适用于任何作物，其中花浸渍方案产生核转基因植株。该项目的目标是通过重组农杆菌将T-DNA传递到叶绿体中，直接转化拟南芥花雌性配子体中的质体。在农杆菌感染期间，VirD2切割T-DNA边界序列，并通过其Tyr29共价连接到t链的5 '端。然后，t链被引导通过IV型分泌系统（T4SS）进入植物细胞，并在那里整合到植物细胞核中。在美国国家科学基金会EAGER基金的支持下，通过去除其核定位信号（NLSs）并将其n端与叶绿体靶向转运肽（TP）融合，对VirD2进行了重组。重组质体VirD2 （Pt-VirD2）在分裂GFP实验中被证明可以定位到叶绿体中，其中VirD2与16个氨基酸的GFP （GFP11）融合，补充了叶绿体中已经存在的一个大的GFP （GFP1-10）。农杆菌介导的叶绿体转化将通过构建同时编码T-DNA和Pt-VirD2功能的载体来实现。Pt-VirD2将引导含有抗生素抗性标记的T-DNA进入质体基因组。为了开发该方法，将通过农杆菌与烟叶共培养和选择载体中编码的抗生素抗性来鉴定转质体事件。然后将拟南芥花浸入携带质体转化载体的农杆菌培养基中，通过在选择性培养基上发芽种子来鉴定经质体转化的拟南芥植株。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

High-frequency plastid transformation in Arabidopsis was achieved by using plants hyper-sensitive to spectinomycin, the selective agent used in chloroplast transformation. The current bottleneck of plastid transformation in Arabidopsis is the difficulty of obtaining fertile plants from transplastomic tissue culture cells. This tissue culture limitation will be overcome by using Agrobacterium to directly transform plastids in the oocytes and identification of transgenic events by germinating seed on selective medium. To achieve this goal, Agrobacterium will be re-engineered to enable T-DNA delivery to the plastids of maternal germline cells. This process will avoid tissue culture and tissue-culture induced genetic variation, thereby greatly simplifying the process of obtaining transplastomic plants. Side-stepping the tissue culture process eliminates the need for specialized expertise to practice plastid transformation in Arabidopsis. Therefore, research proposed here will lead to widespread applications of Arabidopsis plastid genome engineering which, combined with the available extensive genomic resources, will have a major impact on basic science and applications in biotechnology. The new protocol for plastid transformation will be applicable in any crop in which the floral dip protocol yields nuclear transgenic plants. The goal of the project is to directly transform plastids in the female gametocyte in Arabidopsis flowers by re-engineering Agrobacterium for T-DNA delivery to chloroplasts. During Agrobacterium infection, VirD2 nicks the T-DNA border sequences and covalently links to the 5’ end of the T-strand via its Tyr29. The T-strand is then guided through the Type IV secretion system (T4SS) to the plant cell, where it integrates in the plant nucleus. With support from the NSF EAGER Grant, VirD2 was reengineered by removing its nuclear localization signals (NLSs) and fusing its N-terminus with a chloroplast-targeting transit peptide (TP). The reengineered plastid-VirD2 (Pt-VirD2) was shown to localize to chloroplasts in a split GFP assay, in which VirD2 fused with 16 amino-acids of GFP (GFP11) complemented a large GFP (GFP1-10) already in chloroplasts. Agrobacterium-mediated chloroplast transformation will be obtained by constructing vectors which encode both T-DNA and Pt-VirD2 function. Pt-VirD2 will direct T-DNA containing an antibiotic resistance marker to the plastid genome. To develop the method, transplastomic events will be identified by cocultivation of Agrobacterium with tobacco leaves and selecting for antibiotic resistance encoded in the vector. Arabidopsis flowers will then be dipped in an Agrobacterium culture carrying the plastid transformation vector and transplastomic Arabidopsis plants will be identified by germinating seed on a selective medium.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.