外源钙缓解低夜温花生光抑制的分子机理

Peanut distribution and production is restrained seriously by low night temperature stress . As what has been found in the early study, exogenous calcium proved to be remarkably effective in peanut cold resistance improvement, especially for alleviating photoinhibition induced by low night temperature stress. Nevertheless, the molecular mechanism of exogenous calcium alleviating photoinhibition of peanut under low night temperature stress effectively has not been clearly indicated so far. Based on this situation, the cold-sensitive peanut varieties are to be selected as research materials and placed in an artificial climate chamber with imitated low night temperature stress. Then those peanut seedlings shall be regulated by Ca2+ and its inhibitors during initial period of photoinhibition. First of all, RNA-Seq technology shall be adopted to identify the key gene for Calcium/Cold interaction; meanwhile, the scanning and transmission electron microscopy antimonite precipitation method and calcium fluorescence indicator - laser confocal microscopy shall be employed to discuss the response rules of leaf calcium distribution to the regulation of Ca2+ and low night temperature stress. Secondly, several technologies such as gene over-expression, CRISPR gene editing, plant photosynthetic physiology technologies (GFS3000, DUAL-PAM-100, IMAGING-PAM etc., for instance) are to be integrated reasonably to analyze function of the key gene for Calcium/Cold interaction. And the qPCR and GUS technologies shall be applied to discuss tissue expression feature of the key gene for Calcium/Cold interaction; Once again other salts (Mg2+ etc., for instance) are to be used to confirm its specificity when regulated by Ca2+ nutrition. In the end, the molecular mechanism of exogenous calcium alleviating photoinhibition of peanut under low night temperature stress will be indicated, and this will lay the theoretical foundation in fertilizers application (calcium fertilizers, for instance) to improve peanut cold resistance and extending peanut cultivation area.

低夜温严重制约花生地理分布和生产。前期研究证实，钙主要通过缓解低夜温光抑制的途径来提高花生耐冷性。但受钙优先调控的钙/冷互作关键基因及其如何缓解低夜温光抑制的机理尚不清楚。为此，我们利用人工气候室模拟低夜温环境，通过叶面增钙及减钙处理花生苗，在光抑制始发期（IMAGING-PAM检测）精确取样，采用RNA-Seq技术确定钙/冷互作关键基因；同时，结合电镜焦锑酸钾沉淀技术及钙离子荧光指示-激光共聚焦显微技术，探讨叶片钙素分布对钙/冷调控的响应规律；其次将CRISPR基因编辑、基因超表达、光合生理等技术有机结合，鉴定钙/冷互作关键基因的功能；并以qPCR和GUS技术，探讨钙/冷互作关键基因的组织表达特征；最后，采用其它盐类（如镁盐等）来证实钙/冷互作关键基因受钙调控的特异性。最终揭示钙缓解低夜温花生光抑制的分子机理，这将为生产中利用施肥（如钙肥等）来改善花生耐寒性和拓展其栽培区域奠定理论基础。

花生是世界重要的油料和经济作物，在保障我国食用油安全方面具有举足轻重的作用。特别是，基于后疫情时代“双循环”新发展格局下、中国大豆产需缺口加大等背景下，中央明确了油料作物结构调整主要策略之一，就是多措并举发展国内花生生产替代大豆进口。因此，我国油料作物供给（食用油）安全和国际新形势新挑战已然将花生的优化施肥技术与耐冷优质高效生产推到一个前所未有的国家战略安全高度。辽宁是我国重要的优质花生出口基地和产区之一，花生在辽宁省大田作物中其种植面积仅次于玉米和水稻，但是辽宁花生低温冷害普遍存在。研究表明，大多数花生在幼苗期环境温度低于12-16℃时，植株生长缓慢严重时停止生长，叶片会出现脱水、萎蔫，甚至枯死等现象。钙主要通过缓解低夜温光合碳同化障碍的途径来提高花生耐冷性，但外源钙缓解低夜温花生光抑制的分子机理尚待探究。为此，本研究选用前期试验筛选出的冷敏感大果型花生作为试验材料，采用人工气候室精量模拟花生早春不同情景短时（小于12 h）和长时（大于12 h）低夜温胁迫及其常温恢复过程等的环境条件，运用叶面喷施钙离子及其抑制剂，研究外源钙对短时和长时低夜温光合障碍的调控作用；其次，通过测定低温胁迫及恢复后的花生幼苗的生长状况、碳水化合物变化、光合气体交换参数、叶绿体超微结构、活性氧含量以及叶绿素荧光参数及生物电子显微成像等，揭示外源钙离子缓解花生低温光合反馈抑制的预激发效应机制；最后，通过叶面增钙及减钙处理花生苗，在光抑制始发期（IMAGING-PAM检测）精确取样，采用RNA-Seq技术确定钙/冷互作关键基因，并鉴定钙/冷互作关键基因（AhCAS）的功能及其组织表达特征；最后，采用其它盐类（如镁盐等）来证实AhCAS受钙调控的特异性，从而揭示钙缓解低夜温花生光抑制的分子机理，这将为生产中利用施肥（如钙肥等）来改善花生耐寒性和拓展其栽培区域奠定理论基础。