小热激蛋白PtsHSP启动子甲基化在半夏响应高温胁迫中的功能研究

Pinellia ternata is one of the widely used traditional Chinese herbs. The most important abiotic factor that limits the yield of P. ternata is the high temperature stress during growth period. Small heat-shock protein genes (sHSPs) can be induced with a sharp increase by high temperature in plants , thereby enhancing their adaptation to high temperature stress. However, the mechanism by which sHSPs are induced is not yet known. Previously, we confirmed that the transcripts of PtsHSP increased and its promoter appeared demethylation in P. ternata treated with high temperature. Therefore, it is speculated that the methylation status of PtsHSP promoter involved in the regulation of the response to high temperature stress in P. ternata. In this project, we planed to ascertain the regulatory effect of promoter methylation on expression patterns of the promoter and PtsHSP via comparing the methylation patterns of PtsHSP promoter and expression patterns of PtsHSP under high temperature. Followingly, it was aimed to identify core methylation region of the promoter that responses to high temperature stress by promoter fragmenting technique, and the restorer lines should be created via transforming the vectors of PtsHSP:: PtsHSP and mPtsHSP::PtsHSP into PtsHSP knockout lines respectively. Then, the survival rate and the related physiological and biochemical indexes of the above lines under high temperature stress were analyzed, so as to elucidating the mechanism of core methylation region in the promoter in regulating the response of P. ternata to high temperature stress. This study will broaden our understanding of epigenetic aspects on P. ternata responsing to high temperature, further providing reference for the exploration of heat adaption strategy and genetic improvement of P. ternata.

半夏是我国常用的大宗中药材之一，生长中遭遇的高温是限制其产量的关键因素。高温可通过诱导植物体内小热激蛋白基因（sHSPs）的表达，增强植物对高温的适应性，但sHSPs受高温诱导的分子机理尚不知晓。申请者初步研究发现，高温不仅诱导半夏PtsHSP的表达，且导致其启动子去甲基化，推测PtsHSP启动子甲基化参与调控半夏的高温胁迫响应。本项目拟通过分析高温胁迫下PtsHSP的表达模式及其启动子的甲基化模式，明确启动子甲基化在PtsHSP响应高温胁迫中的作用；利用启动子分段技术，明确启动子中响应高温胁迫的核心甲基化区段；获得PtsHSP::PtsHSP及mPtsHSP::PtsHSP转化ptshsp的恢复系，比较二者在高温胁迫下的表型，明确核心甲基化区段在半夏高温胁迫中的功能。本研究从表观遗传方面加深对半夏高温胁迫响应机理的认识，为探索半夏的高温适应策略及品种改良提供了参考依据。

半夏是我国常用的大宗中药材之一，生长中遭遇的高温是限制其产量的关键因素。高温可 .通过诱导植物体内小热激蛋白基因（sHSPs）的表达，增强植物对高温的适应性，但sHSPs受高温诱导的分子机理尚不知晓。本课题围绕高温胁迫过程中PtsHSP启动子甲基化的功能展开研究，主要研究内容包括：高温胁迫下PtsHSP表达模式及启动子甲基化模式分析；PtsHSP启动子中核心甲基化区段的鉴定及功能验证。结果表明，PtsHSP基因表达量在叶片中最高，叶柄次之，块茎中含量最少。PtsHSP基因在半夏叶片中显著受高温诱导上调表达，随胁迫时间延长，该基因表达总体呈持续升高趋势。其启动子区域两段CpG岛的甲基化率均受高温诱导显著下调，随高温处理时间的延长，CpG岛中胞嘧啶甲基化率出现一定的波动，但整体甲基化率相比高温胁迫4 h时的甲基化率无显著变化。通过启动子不同缺失实验，证明启动子中的CpG岛Ⅰ为其核心甲基化区域，参与高温胁迫条件下PtsHSP基因的表达调控，转基因半夏功能分析显示PtsHSP启动子区域的CpG岛Ⅰ参与调控半夏的高温胁迫响应。本研究从表观遗传方面加深对半夏高温胁迫响应机理的认识，为探索半夏的高温适应策略及品种改良提供了参考依据。