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

利用叶绿体转化中使用的选择剂壮观霉素高度敏感的植物，实现了拟南芥的高频叶绿体转化。目前，拟南芥中叶绿体转化的瓶颈是从转体组织培养细胞中获得可育植株的困难。利用农杆菌直接转化卵母细胞中的质体，以及通过在选择的培养基上萌发种子来鉴定转基因事件，将克服这种组织培养的局限性。为了实现这一目标，将对农杆菌进行重新设计，使T-DNA能够输送到母体生殖系细胞的质体中。这一过程将避免组织培养和组织培养引起的遗传变异，从而大大简化获得转基因植株的过程。绕过组织培养过程，就不需要专门的专业知识来实践拟南芥中的叶绿体转化。因此，本文提出的研究将导致拟南芥基因组工程的广泛应用，与现有的丰富基因组资源相结合，将对基础科学和生物技术应用产生重大影响。新的叶绿体转化协议将适用于任何花卉DIP协议产生核转基因植株的作物。该项目的目标是通过重新设计农杆菌来将T-DNA输送到叶绿体中，从而直接转化拟南芥花雌配子体中的质体。在农杆菌感染过程中，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)到达植物细胞，在那里它整合到植物细胞核中。在美国国家科学基金会的支持下，VirD2被重组，去除了其核定位信号(NLSS)，并将其N端与叶绿体靶向转运肽(TP)融合在一起。在裂解GFP实验中，重构体-VirD2(pT-VirD2)被证明定位于叶绿体，其中VirD2与GFP的16个氨基酸(GFP 11)融合，补充了叶绿体中已有的一个大的GFP(GFP 1-10)。通过构建同时编码T-DNA和PT-VirD2功能的载体，可以获得农杆菌介导的叶绿体转化。PT-VirD2将含有抗生素抗性标记的T-DNA定向到叶绿体基因组。为了开发该方法，将通过与烟草叶片共培养农杆菌并选择载体中编码的抗生素抗性来识别转体事件。拟南芥的花朵随后将被浸泡在携带叶绿体转化载体的农杆菌培养中，并将通过在选择性介质上萌发种子来鉴定转质体拟南芥植株。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.