Comparative molecular analysis of metal homeostasis in the Arabidopsis model species A. halleri, a metal hyperaccumulator, and A. thaliana

这个项目旨在扩大我们对植物自然选择的、极端的、复杂的特征背后的分子机制的理解。作为模式性状，通过与近缘关系密切的非耐性非积累植物(主要是拟南芥)的比较，研究了拟南芥中锌和镉的超积累及其相关的锌和镉的超耐性。为了研究候选基因，例如AhHMA3(重金属ATPase3)、AhMTP1(金属耐受性蛋白1)和AhFRD3(铁还原酶缺陷3)的功能，我们结合了两种方法。首先，通过RNA干扰载体对哈勒氏曲霉的稳定转化，使候选基因沉默。其次，将候选基因导入拟南芥。由于几个基因可能分别对金属超积累和超耐性做出重要贡献，因此也将进行转基因拟南芥的杂交，以结合不同候选基因的功能。与密切相关的非蓄积体相比，除了基因拷贝数的扩张外，金属超蓄积体的特征似乎是一些候选基因表达水平的增加，而不是编码蛋白功能的变化。AhHMA4(重金属ATPase4)基因的高表达水平在顺式基因中是特定的。通过启动子分析，我们划定了启动子区域，并确定了控制AhHMA4基因高表达的顺式调控序列，目的是确定这些序列元件招募的转录因子。为了确定导致额外候选基因高表达的突变类型，还进行了哈勒和拟南芥之间的启动子交换实验。

This project aims to expand our understanding of the molecular mechanisms underlying naturally selected, extreme, complex traits in plants. As model traits, zinc and cadmium hyperaccumulation and associated zinc- and cadmium-hypertolerance in Arabidopsis halleri are examined through the comparison with closely related non-tolerant non-accumulator species, primarily A. thaliana. To examine the functions of candidate genes, for example AhHMA3 (Heavy Metal ATPase3), AhMTP1 (Metal Tolerance Protein1) and AhFRD3 (Ferric Reductase Defective3), two approaches are combined. Firstly, candidate genes are silenced through the stable transformation of A. halleri with RNA interference constructs. Secondly, candidate genes are introduced into A. thaliana. As several genes are likely to make significant contributions to metal hyperaccumulation and hypertolerance, respectively, transgenic A. thaliana lines will also be crossed to combine the functions of different candidate genes. Besides gene copy number expansions, increases in expression levels of a number of candidate genes rather than changes in the functions of the encoded proteins appear to be characteristic of metal hyperaccumulators in comparison to closely related non-accumulators. High expression levels of AhHMA4 (Heavy Metal ATPase4) genes are specified in cis. Through promoter analysis we delineate the promoter regions and identify the cis-regulatory sequences governing high expression of AhHMA4 genes, and we aim to identify the transcription factors recruited by these sequence elements. To identify the types of mutations responsible for the high expression of additional candidate genes in A. halleri, promoter swap experiments between A. halleri and A. thaliana are also conducted.