菊花CmMAPK蛋白激酶调控磷吸收利用的分子机理研究

Chrysanthemum is the important ornamental flower in autumn as its variety of shapes and colous. Phosphorus is one of the essential nutrients for plant growth and development. Phosphorus deficiency will not only seriously affect the ornamental properties of chrysanthemum, but also increase the production cost. Therefore, the molecular mechanism of phosphorus regulation is significant for phosphorus high efficient genotypes and new varieties breeding in chrysanthemum. The differentially expressed genes of CmMAPK were excavated from the transcriptome database of CmPht1;2 overexpression lines under Pi-starvation conditions. The characteristics of CmMAPK gene in response to low phosphorus stress were preliminarily revealed through sequence and expression analysis. The phosphorylation sites of CmMAPK will be analyzed, and the key phosphorylation sites will be explored through point mutation, in vitro phosphorylation, and in-gel kinase activity analysis. The mechanism of CmMAPK signal transmission at the post-translational modification level will be explained. The mechanism of CmMAPK response to low phosphorus will be elucidated by genetic transformation. Related results can provide excellent genetic resources for efficient utilization of phosphorus and enrich the molecular mechanism of resistance to phosphorus-starvation in chrysanthemum.

菊花（Chrysanthemum morifolium Ramat.）拥有丰富的花型花色，是秋季重要的观赏花卉。磷是植物生长发育最重要的营养元素之一。缺磷不仅会严重影响菊花的观赏性状，还会增加生产成本。因此，开展菊花磷吸收利用调控的分子机理研究对培育菊花磷高效新品种具有极其重要的意义。本研究拟从低磷处理CmPht1;2转基因‘神马’转录组数据库中挖掘丝裂原活化蛋白激酶CmMAPK差异基因，通过序列和表达特性分析，初步揭示菊花CmMAPK基因响应低磷胁迫的特性。对CmMAPK磷酸化位点进行分析，通过点突变、体外磷酸化、胶内激酶活性分析等技术探讨其关键磷酸化位点，在蛋白翻译后修饰水平解释CmMAPK信号传递的作用机制。利用遗传转化手段，进一步阐明CmMAPK对低磷响应的作用机理。相关研究成果可为菊花磷高效利用品种提供优异基因资源，也可丰富菊花耐低磷响应分子机制。

菊花（Chrysanthemum morifolium Ramat.）是秋季重要的观赏和经济花卉，具有丰富的花型、花色、食用和药用价值。磷（P）是植物生长发育所必需的营养元素。本研究分析了菊花丝裂原活化蛋白激酶（MAPK）级联通路CmMKK5-CmMPK13在低磷（LP）胁迫下的反应。在LP处理2天后，CmMPKK5和CmMPK13均在根系中诱导上调。利用烟草叶片瞬时表达系统中的亚细胞定位分析显示，CmMPKK5和CmMPK13蛋白均定位于整个细胞结构。通过定点突变分析，获得了CmMPK13翻译后修饰（PTM）磷酸化位点的突变体T200A、Y202A和T200A-Y202A（Cmmpk13s）。亚细胞定位分析显示，磷酸化位点的改变并不影响其在细胞中的分布。磷酸化分析显示，CmMPK13和T200A可以被CmMKK5磷酸化或发生自磷酸化。然而，Y202A和T200A-Y202A不能被CmMKK5磷酸化也不能发生自磷酸化。在拟南芥转基因株系（Oe）中，CmMKK5过表达可以回补拟南芥突变体mkk5的生长，在LP胁迫条件下提前开花。在LP和HP条件下，CmMPK13-Oe和T200A-Oe均能使拟南芥突变体mpk13提前开花，而Y202A-Oe和T200A-Y202A-Oe则不能提前开花。在菊花中，CmMPK13-Oe中磷酸盐（Pi，H2PO4-）浓度、根结构、高度和叶重均显著高于WT。磷酸化蛋白质组分析表明，CmMPK13可以通过改变菊花的转录和翻译来促进菊花对磷缺乏的反应。本研究可为菊花磷高效利用品种提供优异基因资源，也可丰富菊花耐低磷响应分子机制。