基于交联质谱技术解析大蕉耐冷相关蛋白MKK2、MAPK3和ICE1的互作网络

Low temperature affects the growth, development, productivity and geographical distribution of plants, especially for bananas (included Cavendish, Dajiao and so on) originated in the tropics. Although they are mainly cultivated in tropical and subtropical regions, cold spells often occur during winter or early spring, which can have serious impact on fruit quality and yield, and also cause in some incidents the destruction of banana plantations. The cold resistance of Dajiao was significantly higher than that of main varieties (Cavendish), and our previous studies showed that the MAPK cascade -ICE1 pathway, especially MKK2, MAPK3 and ICE1, played a critical role in Dajiao cold resistance, however, their interaction regulation mechanism is unclear. This research will take transgenic Dajiao seedlings of overexpressing mCherry-MKK2, mCherry-MAPK3 and mCherry-ICE1 as materials, to identify the direct and indirect interaction proteins of MKK2, MAPK3 and ICE1 under low temperature treatment by the latest high throughput and high sensitivity cross-linking mass spectrometry technology, then the Yeast Two-Hybrid system and Bimolecular Fluorescence Complementation (BiFC) used to verify their interaction, and its biological function will be disclosed by creating overexpress plants or mutated-plants using CRISPR/Cas9 system. The outcome of the proposed research will be helpful for in-depth understanding the molecular networks of cold-resistant process that modulated by MKK2, MAPK3 and ICE1 in Dajiao, but also provide key candidate genes for cold-resistant banana breeding.

低温影响植物的生长发育、产量和生态分布，而对于热带起源的香蕉（包括香牙蕉、大蕉等）来说更是如此。近年来频繁发生的倒春寒已给我国香蕉产业造成了巨大的经济损失。生产实践中发现大蕉的耐冷性显著强于主栽品种香牙蕉，申请人前期的研究表明MAPK级联-ICE1途径，特别是MKK2、MAPK3和ICE1在大蕉耐冷过程中起着关键的作用，然而目前对其互作调控机理尚不清楚。本项目拟以超表达mCherry-MKK2/MAPK3/ICE1转基因大蕉幼苗为材料，采用最新的交联质谱技术高通量和高灵敏度地鉴定低温处理下与大蕉MKK2、MAPK3和ICE1直接和间接互作的蛋白，然后利用酵母双杂和双分子荧光互补实验进行验证，最后通过在香蕉中超表达和突变重要互作蛋白编码基因解析其生物学功能。本项目研究结果不但可以深入揭示MKK2、MAPK3和ICE1在大蕉抗寒过程中的作用机理，也可为香蕉抗寒分子育种提供有效的基因资源。

低温是限制香蕉生长、产量和地理分布的主要环境因素之一。由于香蕉耐寒性状的复杂性，其耐寒性状的分子机制和相关基因网络在很大程度上是未知的。因此，本研究旨在基于转录组和代谢组联合分析、蛋白质互作验证来识别冷胁迫响应基因、代谢物以及代谢途径，以了解冷敏感香牙蕉和耐冷大蕉耐冷性差异的机制。基于代谢组分析，发现冷胁迫下更多脂质在耐寒大蕉中的积累显著高于冷敏感香牙蕉，表明其在保护香蕉免受寒害方面的重要性，此外，通过转录组分析，在香牙蕉冷胁迫下共鉴定出4626个差异表达基因，在大蕉中共鉴定出5516个差异表达基因。通过结合转录组、代谢组和蛋白质互作数据，我们发现非ABA依赖的脂质代谢途径和MAPK信号转导途径(MKK2-MAPK3-ICE1-POD P7)在大蕉的冷胁迫适应中起着至关重要的作用。本研究结果为了解香蕉冷响应的分子基础及耐寒机制提供了新的见解，为香蕉耐寒性的遗传改良提供了潜在的应用。