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

TRTech-PGR：农杆菌介导的质体基因组转化

• 批准号: 2224861
• 负责人: Pal Maliga
• 金额: $ 146.28万
• 依托单位: Rutgers University New Brunswick
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2224861&HistoricalAwards=false
• 关键词: TRTech; PGR; Agrobacterium; mediated; transformation

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.

通过使用对叶绿体转化中使用的选择性剂对Spectinomycin的植物过度敏感性，可以实现拟南芥中的高频质体转化。拟南芥中质体转化的当前瓶颈很难从移植组织培养细胞中获得肥沃的植物。通过使用农杆菌直接在卵母细胞中转化质体并通过在选择性培养基上发芽种子，可以克服这种组织培养的限制。为了实现这一目标，将重新设计农业，以使T-DNA递送至母体种系细胞的塑料。该过程将避免组织培养和组织文化引起的遗传变异，从而大大简化了获得移植植物的过程。侧向组织培养过程消除了对拟南芥中质体转化的专业知识的需求。因此，此处提出的研究将导致拟南芥质体基因组工程的广泛应用，该技术与可用的广泛基因组资源相结合，将对基础科学和生物技术中的应用产生重大影响。新的质体转化方案将适用于花卉浸入协议产生核转基因植物的任何农作物。该项目的目的是通过重新工程农杆菌将T-DNA递送到叶绿体中，直接在拟南芥花中直接转化雌性配子体的质体。在农杆菌感染期间，VIRD2通过其Tyr29划分了T-DNA边界序列，并共价连接到T链的5'端。然后将T链通过IV型分泌系统（T4SS）引导到植物细胞，并在植物核中整合。在NSF急切的赠款的支持下，VIRD2通过去除其核定位信号（NLSS）并将其N末端与叶绿体靶向的转运肽（TP）进行了重新设计。重新设计的PlastID-VIRD2（PT-VIRD2）被证明在分裂的GFP测定中定位于叶绿体，其中Vird2与16个氨基酸含量GFP（GFP11）融合了大型GFP（GFP1-10）在氯植物中已经完成了大型GFP（GFP1-10）。农杆菌介导的叶绿体转化将通过构造编码T-DNA和PT-VIRD2功能的向量来获得。 PT-VIRD2将将含有抗生素抗性标记物的T-DNA引向质体基因组。为了开发该方法，将通过与烟草叶子的农杆培养并选择载体中编码的抗生素耐药性来确定移植事件。然后，拟南芥花将被浸入带有质体转化载体的农杆菌培养物中，并且可以通过在选择性媒介上发芽种子来识别移植拟南芥植物。该奖项反映了NSF的法定任务，并通过使用该基金会的知识分子优点和广泛的影响来评估NSF的法定任务，并通过评估诚实地表示支持。