匍匐翦股颖小热激蛋白基因AsHSP26.8应答高温及其他非生物胁迫的功能解析

Creeping bentgrass (Agrostis stolonifera L.) is a common perennial cool-season turfgrass, which has excellent characteristic of cold, salt and shade resistance. It can form a graceful landscape lawn and has been widely used for golf courses and grass court due to its quick turf-establishment and good texture. However, it experiences “summer-kill” in warm and transition area which reduces the landscape and quality of summer turf due to its poor heat resistance. Currently, the molecular mechanisms of creeping bentgrass heat resistance and heat responsiveness are still unclear and therefore, becoming a research hotspot for turfgrass investigators. In this project, we will clone a small heat shock protein gene, AsHSP26.8, from creeping bentgrass based on previous data. The expression profiling of this gene in different tissue under stress conditions and by plant hormones will be determined. For subcellular localization analysis, we will transform the according plasmids into rice leaf via transient transformation. The reverse genetics approach will be used in this project to analyze the function of AsHSP26.8 gene. Heat and other abiotic stress tolerance tests will be screened for transgenic creeping bentgrass plants and wild type. We will further screen interacting gene of AsHSP26.8 based on RNA-seq data. The detailed function of interacting genes will be further characterized. This project will illuminate the detailed functions of AsHSP26.8 during the response of creeping bentgrass to abiotic stresses.

匍匐翦股颖是一种重要的冷季型草坪草，具有质地好，抗逆性强等特点，但是抗热性较差。因此，挖掘匍匐翦股颖热胁迫关键基因是了解匍匐翦股颖热胁迫响应途径的重要手段。本项目基于RACE技术获得匍匐翦股颖小热激蛋白AsHSP26.8基因全长序列，对其进行生物信息学分析，结合亚细胞定位对其进行归类；建立该基因在不同组织器官和不同胁迫条件下的表达模型并分析该基因启动子的活性；构建该基因的过表达载体和RNAi载体，转化匍匐翦股颖，观察该基因对匍匐翦股颖形态、生长发育和抗逆性的影响；分析该基因对转基因匍匐翦股颖内源基因表达的影响，从而全面深入揭示AsHSP26.8的生物学功能及其所参与的非生物胁迫响应途径中所发挥的作用。

匍匐翦股颖（Agrostis stolonifera L.）是一种常见的多年生冷季型草坪草。它具有耐寒、耐盐、耐阴等优良特性。此外，其成坪速度快，质地良好，能够形成外观非常优美的草坪。但由于其耐热性差，“夏枯”现象广泛存在，严重破坏了其草坪外观和质量，限制了其在气候温暖地区的广泛使用。而目前针对匍匐翦股颖耐热性机理以及对高温胁迫下的响应途径还尚不明确，因此这方面的研究成为了草坪研究者所关注的热点。.本项目通过RT-PCR的方法从匍匐翦股颖中克隆出小热激蛋白基因AsHSP26.8及其启动子，通过生物信息学分析，转基因技术等对其在植株对高温以及其他环境胁迫的响应中的功能与作用进行了深入的研究，其结论如下：.AsHSP26.8开放阅读框为732bp，编码243个氨基酸。其氨基酸序列中含有一个典型的Hsp23结构域/α-crystallin结构域（ACD）。该序列还包括一个叶绿体靶信号肽，一个甲硫氨酸富集区和两个保守区域。通过对其进行生物信息学分析和亚细胞定位分析显示AsHSP26.8属于叶绿体小热激蛋白。AsHSP26.8基因能够被高温胁迫强烈诱导，而其他环境胁迫均不能诱导AsHSP26.8基因的表达。对其启动子进行分析发现其含有HSE、AT-rich element和TATA等热胁迫相关的顺式元件。此外，GUS组织染色表明AsHSP26.8基因启动子能够被高温胁迫诱导激活。异源过表达AsHSP26.8能够使转基因拟南芥对高温，盐或ABA处理更为敏感。而在匍匐翦股颖中过表达AsHSP26.8则抑制了转基因植株根系及地上部分的生长发育，并且使转基因匍匐翦股颖的耐热性和耐盐性有所下降，但抗旱性有所提升。RNA-seq结果表明，AsHSP26.8通过调控一些生长素相关基因以及胁迫相关基因的表达参与生长素、ABA-dependent和-independent等多条信号途径，进而影响了转基因植株的生长发育和胁迫响应。