耐旱蓝藻发菜WSPA蛋白对胞外多糖胶鞘结构稳定性的调控作用研究

Terrestrial cyanobacterium of Nostoc flagelliforme, commonly known as “Facai”, is distributed in arid steppes of northwest China. There its thallus undergoes periodic rewetting and desiccation cycles, and correspondingly the thallus expands and contracts acutely in diameter. Ultrastructural observation revealed that the extracellular polysaccharide (EPS) matrix expands to be grids when rehydrated, while it shrinks with cells being embedded in protective voids when desiccated. Thus the stable alteration of elasticity and rigidity of EPS matrix should play critical protection for cell growth and drought resistance. However, the molecular mechanism underlying this regulation is unclear. WSPA protein was found to be the most abundant protein in EPS matrix of N. flagelliforme; biochemical analysis revealed it has beta-galactosidase activity, and in vitro experiment found that it is correlated to the dynamic regulation of the structure of EPS matrix. Based on these backgrounds, this project intends to explore the concrete roles and mechanisms of WSPA protein in maintaining structural stability of EPS matrix from several aspects, including: To investigate potential transglycosylation activity of WSPA and the affecting factors; With the help of genetic means etc., to clarify the detailed processes and features of WSPA in shaping the structure of EPS matrix during expansion and contraction; To investigate potential synergistic functions of other important matricial components with WSPA in EPS matrix. This research will help gain more insights into drought adaptation mechanisms of N. flagelliforme in arid environments and also provide useful guidance for future application of this species in the improvement of desert ecosystem.

发状念珠藻，俗称发菜，生长于我国西北荒漠地带，对干旱逆境具有极强的适应性。发菜在经受干湿交替变化时，其原植体发生剧烈的收缩和膨胀，电镜观察发现，复水时发菜胞外多糖膨胀成网格状，干燥时细胞被保护于收缩多糖的孔隙中，因此胞外多糖结构的弹性和刚性的稳定调节对发菜细胞的生长和耐旱形成了重要保护，但其调节的分子机制还不清楚。WSPA蛋白是发菜胞外多糖胶鞘中的最丰富蛋白，前期研究揭示WSPA具有β-半乳糖苷酶的水解活性、并与多糖胶鞘结构的动态调节有关。本项目拟从多个层面阐释发菜WSPA蛋白对多糖胶鞘结构稳定性的调节功能与机制，包括：WSPA的转糖苷合成活性及各种影响因素的分析；借助遗传手段等重点探究WSPA在发菜“膨胀与收缩”中对多糖胶鞘结构调节的理化特征变化；分析胶鞘中其它重要组分与WSPA的互作与协同作用。本项目研究将拓深对发菜适应环境逆境机制的认识，并将为利用发菜改良荒漠生态提供一定的理论指导。

陆生蓝藻发菜对荒漠地区的干湿交替胁迫或周期性干旱具有极强的适应性。发菜胞外多糖基质起着重要的生理生态的保护性作用，胞外多糖在复水和失水时会相应地引起发菜膨胀和收缩，会对细胞造成潜在的机械胁迫伤害，但这并未发生，因此胞外多糖结构稳定性的有机调控是一个重要的科学问题。本项目研究从发菜胞外最丰富蛋白WspA入手，探究其基因转录特点，解析其生化本质和特性以及对胞外多糖结构动态的调控机制。转录分析发现，该基因受干旱、低温、渗透、盐离子和紫外胁迫诱导表达，其中紫外胁迫诱导效果最为明显，但发菜在复水条件下wspA基因的诱导转录受到抑制。通过体外分段表达和生化分析，发现WspA是一种全新的β-半乳糖苷酶，具有糖苷水解和转糖苷合成作用，确定了其最佳酶活条件（最佳温度45℃，最佳pH为8.0）和中心酶活性区域（基因的562-902碱基，114个氨基酸区域）等；此外，通过分子筛实验，确定该酶蛋白是一种二聚体蛋白，其聚合状态不受pH影响。利用WspA溶液处理发菜，能导致多糖松散和藻丝断裂，发菜藻丝的弹性和抗旱性均下降。液体培养发菜中检测不到WspA蛋白，且不耐脱水胁迫，但在固体平板上适度干旱培养，能逐渐诱导WspA合成、并使得培养发菜获得一定抗脱水胁迫能力，添加额外WspA蛋白情况下，培养发菜能生成更多的胞外多糖且多糖的结构特性也发生了改变，发菜抗脱水能力也更强。发菜的胞外多糖基质中还含有紫外吸收色素―类菌胞素氨基酸（MAAs）和伪枝藻素，通过两两孵育后的蛋白电泳实验表明伪枝藻素能与WspA蛋白结合，通过等温滴定量热法测试发现MAAs与WspA存在强相互作用。此外，发现胞外多糖对WspA的糖苷酶活性具有促进作用，而两种色素分子对其酶活性均具有抑制作用。通过以上主要实验结果以及相关信息，我们提出了WspA的作用模式，即在发菜复水膨胀过程中，WspA辅助多糖糖链的断裂、增强弹性，防止细胞受到牵扯伤害，而在发菜失水收缩过程中，WspA辅助多糖糖链的交联、增强刚性，防止细胞受到挤压伤害，在此过程中，其酶活性也受到其它胞外组分如MAAs和伪枝藻素的调节。