激酶MhCDPK1磷酸化转录因子MhNAC6调控苹果砧木铝胁迫响应的分子机制

Modern orchard with high investment and unreasonable fertilization lead to seriously soil acidification in apple production. Aluminum toxicity caused by soil acidification is the main factor that limits the growth of apple trees. However, the signal transduction pathway of aluminum stress in apple is still unknown. In my previous research, a key aluminum responsive transcription factor MhNAC6 was screened from apple rootstock Malus hupehensis Rehd. by RNA-seq and plant transgenic approaches, and kinase MhCDPK1, which interacted with MhNAC6, was also identified by CoIP-MS. In this project, the following researches will be conducted. Firstly, the function of MhCDPK1 under aluminum stress will be revealed by transgenic apple plants. Secondly, the signal transduction pathway of MhNAC6 phosphorylated by MhCDPK1 will be uncovered through in vivo and in vitro phosphorylation experiments. Finally, the effect of MhNAC6 phosphorylation on its transcriptional activity and the function under aluminum stress will be explored by phosphorylation site mutation. The results of this study will be significant to elucidate the molecular mechanism of aluminum stress signal transduction to the transcription factor MhNAC6 mediated by apple kinase MhCDPK1. It can also provide an important theoretical basis for breeding apple rootstocks with aluminum-tolerance.

高投入高产出的现代果园生产模式和不合理的施肥措施导致苹果产区土壤酸化现象日趋严重，而因土壤酸化引起的铝毒效应是限制果树生长的最主要因素。目前，关于苹果响应铝胁迫的信号转导途径仍不清楚。申请人前期通过RNA-seq和转基因途径从苹果耐铝砧木平邑甜茶中筛选出响应铝胁迫的关键转录因子MhNAC6，并通过CoIP-MS鉴定出和MhNAC6互作的激酶MhCDPK1。本项目将在此基础上进行以下研究：（1）通过苹果转基因途径研究揭示MhCDPK1在铝胁迫下的功能；（2）通过体内、外磷酸化试验研究揭示MhCDPK1磷酸化MhNAC6的信号通路；（3）通过点突变磷酸化位点试验研究揭示MhNAC6的磷酸化对其转录活性及在铝胁迫下功能的影响。该项研究结果对解析苹果激酶MhCDPK1介导的铝胁迫信号传递给转录因子MhNAC6的分子机制具有重要意义，可为通过分子育种途径培育耐铝苹果砧木提供重要的理论依据。

苹果是我国栽培面积最大的果树之一，果树生长需要合适的土壤pH。近年来，不合理的施肥和管理措施导致苹果产区土壤酸化现象日趋严重。在酸性土壤中，铝毒被认为是限制果树生长发育的最主要因素。在土壤pH<5的果园中，铝以Al3+的形式释放到土壤溶液中被果树根系吸收，高浓度的Al3+会造成果树根系中毒，形成“短而粗硬且不分支”的根系，引发果树营养障碍、生长发育不良以及果实产量、品质下降等问题。但是目前关于苹果响应铝胁迫的分子机制以及信号传导研究尚不清晰。本研究在前期研究基础上通过转录组测序和表达量分析出关键的激酶MhCDPK1和转录因子MhNAC6，并通过CO-IP，BIFC以及酵母双杂试验发现他们具有互作关系，并发现MhCDPK1能够磷酸化MhNAC6进而调控下游基因。转基因MhNAC6植株表现较好耐铝性，同时发现，过表达MhNAC6植株中的氢离子从细胞中转运至细胞间隙，完成离子交换，改善细胞内的PH值，从而提高植物的耐铝性。此外，我们还发现转基因MhCDPK1植株还表现出液泡与细胞质间的氢离子和铝离子交换，这是转基因MhNAC6植株不具有的表现。综上所述，研究表明MhCDPK1磷酸化MhNAC6完成离子交换和PH值调节。本研究揭示在苹果中“铝胁迫信号”，通过“MhCDPK1磷酸化信号”传递给“转录因子MhNAC6”，改变其转录活性以响应铝胁迫的分子机制。该项研究对于解析MhCDPK1介导的苹果铝胁迫信号转导途径具有重要意义，也为通过分子育种途径培育耐铝苹果砧木提供重要的理论依据和基因资源。