构树BpMYB1基因调控镉胁迫响应的功能机理研究

Broussonetia papyrifera is an economic tree with important environmental value. It has strong resistance to heavy metal stress and is an ideal material for heavy metal research. However, there were few reports about the mechanism of resistance to heavy metal stress in B. papyrifera. MYB is a very important transcription factor family for plant responses to stress. In the early study of resistance to heavy metal stress, we found that the MYB transcription factors from B. papyrifera can be induced by cadmium stress, and MYB transgenic yeasts can effectively improve the ability to cadmium stress. Therefore, in this study, a MYB gene (named as BpMYB1), which was most significantly induced by cadmium stress, will be chosen to construct overexpression and RNAi-silence vector and transformed into wild type B. papyrifera, respectively. The functions on cadmium resistance and regulating to defence genes of downstream will be clarified. The expression difference of downstream target genes among overexpression, RNAi-silence lines and WT plant will be compared by chip technology, the downstream target genes regulated by BpMYB1 and the regulatory network they participantwill be determined. The cis-acting elements interacted with BpMYB1 will studied by chromatin immunoprecipitation (ChIP) method, and their interaction intensity will be verified by yeast one-hybrid method, then the mechanism of cadmium regulation of BpMYB1 gene will be elucidated. This study will not only generate a foundation for further study the adaptation mechanism to heavy metals, but also provide strong support for developing the suitable area of B. papyrifera.

构树是具有重要环境价值的经济树种，对重金属胁迫具有极强的抗性，是进行抗重金属研究的理想材料。然而，迄今关于构树抵抗重金属胁迫调控机制方面的研究鲜见报道。MYB是植物响应逆境的一类非常重要的转录因子。在前期对构树抗重金属胁迫研究中，我们发现MYB类转录因子能被镉胁迫明显诱导，转MYB基因的酵母能有效的提高抗镉胁迫能力。因此，本研究选取其中一条被镉胁迫诱导表达最为明显的MYB基因（BpMYB1）构建过表达和抑制表达载体并分别转入野生构树，研究其抗镉能力及对下游防卫反应相关基因的表达调控。用芯片技术比较转BpMYB1基因构树与野生型构树的基因表达差异，确定BpMYB1所调控的下游靶基因及抗逆调控网络。用染色质免疫共沉（ChIP）方法研究BpMYB1识别的DNA元件，用酵母单杂交等方法验证其互作强度。通过本研究阐述BpMYB1基因的抗镉调控机制，为揭示构树抵抗重金属的适应机制奠定基础。

构树是具有重要环境价值的经济树种，对重金属胁迫具有极强的抗性，是进行抗重金属研究的理想材料。然而，迄今关于构树抵抗重金属胁迫调控机制方面的研究鲜见报道。MYB是植物响应逆境的一类非常重要的转录因子。研究选择构树BpMYB1基因，构建过表达和抑制表达载体并分别转入野生构树，研究其抗镉能力及对下游防卫反应相关基因的表达调控。用芯片技术比较转BpMYB1基因构树与野生型构树的基因表达差异，确定BpMYB1所调控的下游靶基因及抗逆调控网络。用染色质免疫共沉（ChIP）方法研究BpMYB1识别的DNA元件，用酵母单杂交方法验证其互作强度。研究比较BpMYB1基因过表达和抑制表达转基因构树与野生型构树在形态、生理方面的差异，确定了BpMYB1基因对Cd的吸收和耐受性正调节功能。明确了其下游调控中涉及的防卫反应相关基因（SOD、POD、GST、PATPase、V-ATPase、HSP70、HSF、WRKY、NAC、Dof、GRAS等）及其参与的调控途径（GA信号、ROS代谢等）。得知BpMYB1可识别MYBCORE、TATCCAOSAMY元件；BpMYB1可直接识别BpFC2、BpbHLH93、BpbZIP1、BpbZIP-CPC1等相关基因参与Cd响应。考虑到GA的应用促进了BpMYB1的表达和Cd积累或耐受性，研究总结出基于BpMYB1和下游目标之间的识别的反馈机制控制植物细胞中Cd的吸收和积累的调控机制。本研究的结果使我们能够理解植物通过BpMYB1和GA信号对Cd胁迫响应的潜在机制，为创制具有增加或减少Cd吸收量的转基因植物在Cd污染土壤中生长甚至从Cd污染地区去除Cd提供了思路。