苯丙烷代谢反馈调控相关转录因子CwMYB2在马蹄黄化过程中的功能及作用机理研究

Surface etiolation is the main limiting factors of fresh-cut Chinese water chestnut (CWC) quality, because of the mechanism of fresh-cut CWC had not been completely elucidate, at present, effective control technique of fresh-cut CWC etiolation had not been developed. Our previous study found fresh-cut CWC etiolation was closely related to phenylpropanoid metabolism, and phenylpropanoid metabolic intermediates could delay fresh-cut CWC etiolation by suppressing the expression of key enzyme coding genes in phenylpropanoid metabolism. The MYB transcription factors are key regulators in phenylpropanoid pathway, forty-seven Unigene sequences that share high homology with MYB protein were obtained from the floral transcriptome database of CWC. Through a preliminary analysis, we presume that CwMYB2 might play an important role in feedback regulation of phenylpropanoid pathway. In this study, taking CWC ‘Fang Lin’ cultivar (a leading variety) as research material, the present proposal aimed to identify its function by analyzing the expression pattern of the CwMYB2 and its subcellular localization; to elucidate the mechanism of transcriptional regulation of CwMYB2 by analyzing the interaction effect between CwMYB2 with other transcription factors and the Transcriptional activation activity of CwMYB2 against the promotor of the structure genes in phenylpropanoid metabolism to analyze the function of CwMYB2 by obtaining the overexpressed or interfered transgenic plants. This project will help us understand the mechanism of the feedback regulation of phenylpropanoid metabolism, also provide theoretical basis and new ideas of the prevention and control measures for CWC etiolation.

表面黄化是限制鲜切马蹄品质的主要因素，由于马蹄黄化机理尚未完全阐明，目前仍未开发出有效的鲜切马蹄黄化控制方法。我们前期工作发现苯丙烷代谢与马蹄黄化密切相关，通过代谢中间产物反馈抑制苯丙烷途径关键基因的表达能有效控制马蹄黄化。MYB是调控苯丙烷代谢的关键转录因子，前期在马蹄转录组中发现了47个MYB转录因子成员，通过初步表达分析推测CwMYB2可能在马蹄苯丙烷代谢的反馈调节中起着重要作用。本项目拟以‘芳林’马蹄为试材，通过分析CwMYB2在不同组织的表达特征和亚细胞定位，初步阐明CwMYB2的表达特性；通过分析CwMYB2与其它转录因子的互作和对苯丙烷代谢结构基因启动子的激活活性，阐明其转录调控机制；并在马蹄和烟草叶片中过量和干涉CwMYB2表达，探析其在苯丙烷代谢反馈调控中的功能。本项目的研究有助于我们深入理解植物苯丙烷代谢的反馈调控机理，也为鲜切马蹄黄化的防控提供理论依据和新思路。

鲜切马蹄贮藏过程中表面极易黄化，但马蹄球茎的黄化与传统果蔬酶促褐变存在差异，其分子调控机制尚未完全解析。由于缺少有效的抑制技术，黄化现象严重影响了鲜切马蹄产业的发展。最近研究发现，鲜切马蹄黄化的主要原因是球茎伤口处黄酮类物质的大量积累。因此，控制鲜切马蹄的黄酮代谢是抑制其黄化的关键。我们研究发现苯丙烷代谢中间产物阿魏酸、肉桂酸和香豆酸等能有效抑制马蹄黄化，表明植物黄酮代谢不但受到各种生理及环境因素的调控，还受该途径自身次生代谢中间产物的反馈调节。本项目利用二代和三代转录组测序筛选出差异表达的黄酮合成相关转录因子CwMYB2。通过表达分析发现马蹄去皮后CwMYB2的表达快速提高，其表达变化与马蹄黄化程度和黄酮类物质的积累呈正相关，并且在马蹄茎和根部也有较高的表达水平。通过亚细胞定位分析发现CwMYB2和其它差异表达R2R3-MYBs均定位于细胞核。利用染色体步移法获得CwPAL、CwCHS等黄酮合成关键结构基因的启动子序列，均发现有MYB 结合位点。验证了CwMYB2对黄酮合成相关结构基因启动子的激活活性，表明CwMYB2能够激活马蹄中黄酮合成相关基因的表达。在烟草叶片中过表达CwMYB2发现其能够提高烟草黄酮代谢及其上游莽草酸合成途径关键结构基因的表达，并使烟草叶片中过量积累二氢黄酮和黄酮醇类物质。构建CwMYB2过表达载体，通过农杆菌转染获得转基因马蹄苗，进一步分析发现CwMYB2的过表达能够显著影响马蹄叶片中的黄酮含量。除了苯丙烷代谢中间产物，茉莉酸信号抑制剂处理也能抑制CwMYB2的表达并且延缓马蹄黄化。本项目研究结果表明苯丙烷代谢中间产物处理能显著抑制CwMYB2的表达从而抑制马蹄黄酮类物质的积累。CwMYB2在马蹄黄酮合成过程中起重要的调控作用，其表达能快速响应机械伤信号进而促进组织细胞中相关基因的表达和黄酮类物质的合成。