高温胁迫下半夏硬脂酸-ACP去饱和酶基因的表达与功能研究

Pinellia ternata is a widely used traditional Chinese herb. However, the wild and artificially cultivated production can just meet 1/3 of market demand. The most important abiotic factor is the high temperature stress during growth period. It has been broadly confirmed that the change in stearic acid-ACP desaturase gene (SAD) expression resulted from temperature stress is involved in the regulation of fatty acid saturation degree, further affecting the tolerance of plant to low temperature. However, the related studies are rarely focused on the relationship between SAD gene expression and high temperature, not to mention the SAD gene of P. ternata in response to temperature. Previously, we confirmed that expression level of Pinellia ternata stearic acid-ACP desaturase gene (PtSAD) changed remarkably under heat stress, indicating that PtSAD may take a key role in response high temperature stress. Employing PtSAD as study object, we planned to investigate the high temperature- inducible expression pattern and underlying mechanism by promote isolation and expression pattern, and to generate the PtSAD-overexpressing and knocked-out materials for analyzing the phenotype and fatty acids composition. Furthermore, the membrane integrity, protective enzymes and membrane lipid peroxidation were precisely measured, so as to elucidating the physiological basis of PtSAD in response to high temperature. This study will broaden our understanding on P. ternata to high temperature stress, further providing reference for the exploration of heat adaptation strategy and genetic improvement of P. ternata.

半夏为一种常用的大宗中药材，目前野生和人工栽培产量仅能满足市场需求量的1/3，其中最重要的限制因素为生长中遭遇的高温逆境。大量研究表明，高温环境下硬脂酸-ACP去饱和酶（SAD）参与调节膜脂肪酸饱和程度，进而影响植物对温度的耐受性。但迄今尚无半夏硬脂酸-ACP去饱和酶基因（PtSAD）功能的公开报道。项目组发现PtSAD在高温环境下表达水平发生显著变化。我们推测其在半夏响应高温胁迫过程中起重要作用。本项目拟以PtSAD为研究对象，通过启动子分离和表达模式鉴定，研究其应答高温的诱导模式和潜在机理；同时创制超表达及敲除突变材料，分析高温胁迫表型及基因表达变化，并测定脂肪酸组成。在此基础上，利用生理生化手段，精确测定膜完整性、膜保护酶活性及膜脂过氧化程度等参数，阐明PtSAD在高温胁迫下发挥作用的生理基础。本研究将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参考依据。

针对高温逆境引发的“倒苗”严重影响半夏生产的问题，项目组在前期通过抑制性消减杂交挖掘高温与常温处理下差异表达基因的基础上，开展了硬脂酸-ACP去饱和酶基因（PtSAD）的功能研究。本项目通过启动子分离和表达模式鉴定，研究其应答高温的诱导模式和潜在机理；创制超表达及敲除突变材料，分析高温胁迫下半夏植株表型变化和脂肪酸组成，测定膜完整性、膜保护酶活性及膜脂过氧化程度等生理生化指标。CRISPR-cas9创制的PtSAD突变体半夏植株耐热性增强，而35S启动子驱动PtSAD基因超表达后植株耐热性降低，表明PtSAD负向调控半夏耐热性。与对照植株相比较，敲除突变体十八烷酸（18:0）含量增加了13.2%、油酸含量降低20.4%，而超表达植株中18:0降低8.6%，油酸含量增加17.4%，说明PtSAD直接参与调控半夏细胞膜脂肪酸组成。生理生化分析表明，敲除PtSAD有助于维持植株在高温下细胞膜完整性，保持抗氧化酶活性和抑制膜脂过氧化；而过表达后则细胞膜完整性被破坏，抗氧化酶活性受抑制，膜脂过氧化程度加剧。qRT-PCR分析发现其表达量在高温胁迫下增加15倍；采用Hi-TAIL 技术，成功克隆PtSAD基因其实密码子上游1756 bp调控区域，通过生物信息学分析1239 bp处存在与胁迫响应相关的TC-rich repeats（GTTTTCTTAC）元件，提示TC-rich repeats可能参与调控半夏PtSAD对高温胁迫的响应。综上，本项目证实了PtSAD通过负向调节半夏细胞膜脂肪酸组成影响植株耐热性，其启动子中TC-rich repeats可能直接调控高温响应，高温胁迫下PtSAD敲除突变体能维持细胞的生理生化指标稳定，使得半夏植株能经受高温逆境胁迫。该研究结果有助于探索半夏的高温适应策略及半夏耐热品种的遗传改良。