Molecular analysis of activation tagged aspen-Populus variants II

激活标记白杨-杨变体 II 的分子分析

• 批准号: 254171702
• 负责人: Privatdozent Dr. Matthias Fladung
• 金额: --
• 依托单位: Institut für Forstgenetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/254171702?language=en
• 关键词: Molecular; analysis; activation; tagged; aspen; Molecular; analysis; activation; tagged; aspen

So far, only very few naturally available tree mutants are known, most of them showing alterations in tree habitus (e.g. dwarf, columnar, pendula mutants) and/or variations in leaf form/shape/color (e.g. lanceolate, brevifolia, and pale green leaf mutants. In two preceding proposals, the fundamentals for transposon (Ac from maize) based activation tagging in a tree species (Populus spec.) were developed. Proof-of-concept for using this system to generate poplar mutants was already provided. In this proposal, we are aiming to consider highly innovative aspects, namely (a) to investigate already available poplar variants obtained in the preceding project and characterize the genes involved in the mutation by over-expressing of the candidate genes in poplar, (b) to produce new mutants by employing a novel and efficient tagging strategy, and (c) to test different possibilities for the long-term storage of mutant plants and mutant tissue (incl. cryopreservation) of a tree species. Characterization of the already available hybrid aspen variants will be done as following: (a) all putative activation tagged variants still available will be further investigated molecularly as well physiologically, (b) for many of the variants, a candidate gene was identified putatively responsible for the variant phenotype: the gene of up to 5 most interesting variants will be cloned into a binary vector and over-expressed in hybrid poplar through genetic transformation. We will be able to prove whether the candidate gene is responsible for the variant phenotype latest in the third year of the project. Compiling all the information obtained, we will develop a vision of what to do with the variants and how to save all the lines produced to build up a collection of poplar variants. As a revised tagging strategy, we will establish a functional ATDs-tms-based transposon approach to produce plants with high probability of transposition (being putative variants). For this, first, the NAM concentration in the regeneration medium has to be optimized, and second the number of different transgenic lines will be enlarged to eliminate transposition-silenced lines at an early stage of the experimental procedure. For long-term storage of variants, at first regenerative callus cultures will be established from the mutant lines and be kept at low temperature. Also re-cutting the mutant plants during glasshouse culture will be tested. In cooperation with a French lab, cryopreservation of the regenerative callus cultures in liquid nitrogen will be tested. In the third year, the stability of mutant phenotypes will be tested.

So far, only very few naturally available tree mutants are known, most of them showing alterations in tree habitus (e.g. dwarf, columnar, pendula mutants) and/or variations in leaf form/shape/color (e.g. lanceolate, brevifolia, and pale green leaf mutants. In two preceding proposals, the fundamentals for transposon (Ac from maize) based activation tagging in a tree species (Populus spec.) were开发的概念。测试不同的可能性的突变植物和突变组织（包括冷冻保存）的可能性。多达5种最有趣的变体将通过遗传转化克隆到二元载体中，并在混合杨树中过表达。我们将能够证明候选基因是否负责该项目第三年的最新变体表型。汇编所有获得的信息，我们将建立一个愿景，了解如何处理变体，以及如何保存所有生产的行以建立杨树变体的集合。作为修订的标记策略，我们将建立一种基于ATDS-TMS的功能性转座子方法，以生产具有较高换位可能性（假定变体）的植物。首先，必须优化再生培养基中的NAM浓度，其次，将在实验过程的早期阶段将不同的转基因线的数量扩大以消除转置脱水线。为了长期存储变体，首先将从突变线建立再生愈伤组织培养物，并保持在低温。还将测试在温室培养过程中重新切割突变植物。与法国实验室合作，将测试对液氮再生愈伤组织培养物的冷冻保存。在第三年，将测试突变表型的稳定性。