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Molecular analysis of activation tagged aspen-Populus variants II

激活标记白杨-杨变体 II 的分子分析

基本信息

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

项目摘要

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.

到目前为止，只有极少数天然可用的树木突变体是已知的，其中大多数表现出树木习性的改变（例如矮化、柱状、钟摆突变体）和/或叶形/形状/颜色的变化（例如披针形、短叶和淡绿叶突变体。在前面的两个提案中，树种（杨属规格）中基于转座子（来自玉米的Ac）激活标记的基础是开发的。已经提供了使用该系统生成杨树突变体的概念验证。在本提案中，我们的目标是考虑高度创新的方面，即（a）研究在先前项目中获得的现有杨树变种，并通过在杨树中过度表达候选基因来表征参与突变的基因，（b）通过采用新颖且有效的标记策略来产生新的突变体，以及（c）测试长期储存树种突变植物和突变组织（包括冷冻保存）的不同可能性。已经可用的杂交白杨变体的表征将按如下方式进行：（a）所有仍然可用的推定的激活标记变体将在分子和生理学上进一步研究，（b）对于许多变体，鉴定出推定负责变体表型的候选基因：多达 5 个最有趣的变体将被克隆到双元载体中，并通过遗传转化在杂交杨中过度表达。最迟在项目的第三年，我们将能够证明候选基因是否与变异表型有关。汇总获得的所有信息，我们将制定如何处理变体以及如何保存生成的所有品系以建立杨树变体集合的愿景。作为修订后的标签策略，我们将建立一种基于 ATDs-tms 的功能性转座子方法，以生产具有高转座概率（假定变体）的植物。为此，首先必须优化再生培养基中的NAM浓度，其次要扩大不同转基因株系的数量，以在实验过程的早期阶段消除转座沉默株系。为了长期储存变体，首先将再生愈伤组织培养物从突变系建立并保持在低温下。还将测试在温室培养期间重新切割突变植物。将与法国实验室合作，测试液氮中再生愈伤组织培养物的冷冻保存。第三年，将测试突变表型的稳定性。