甘蔗响应宿根矮化病菌致病因子pglA的关键基因SnRK1β1的克隆及功能解析

由Leifsonia xyli subsp. xyli (Lxx)引起的宿根矮化病（RSD）严重影响甘蔗产量和宿根性，是植蔗区重要的甘蔗病害。pglA是Lxx的关键致病因子，具有溶解细胞壁的能力。SnRK1家族基因在植物应答病原菌入侵中起关键作用，前期基于酵母双杂交研究证实甘蔗SnRK1β1基因与pglA发生互作。为了解SnRK1β1基因表达特性及功能，本项目拟在分离到该基因片段的基础上，利用同源克隆结合RACE技术扩增基因全长并进行生物信息学分析；定量PCR分析基因在不同抗性品种、不同接种时期和非生物胁迫下的基因表达特性；构建基因过量表达和RNAi干扰载体，把过量载体导入烟草和甘蔗，RNAi载体导回甘蔗，对转基因植株的农艺性状、基因表达、RSD抗性、磷酸化蛋白质组和SnRK1酶活等进行分析，以期揭示甘蔗SnRK1β1基因在响应Lxx侵染过程中的功能，为甘蔗RSD的抗病分子育种提供新理论。

Sugarcane ratoon stunting disease, caused by Leifsonia xyli subsp. xyli (Lxx), is an important disease in sugarcane planting regions. It is a key factor affecting the yield and ratoon of sugarcane. pglA is the key pathogenic factor of Lxx and it has the ability to dissolve cell wall. SnRK1 family genes play a key role in plant response to pathogen invasion. It was found that SnRK1β1 gene from sugarcane interacted with pglA by yeast two-hybrid technique. In order to illuminate the expression characteristics and functions of SnRK1β1, this research protocol is going to clone full-length sequence of SnRK1β1 from sugarcane (Saccharum officinarum) by homology cloning strategy and rapid-amplification of cDNA ends, and analyze them with relative bioinformatics method. The gene expression of SnRK1β1 will be analyzed using Real-time fluorescent quantitative PCR in different sugarcane varieties, different infection period and under various abiotic stresses. The over expression vector will be constructed and be introduced into tobacco and sugarcane, also RNAi vector will be constructed and be introduced into sugarcane. The agronomy traits, genes expressions, RSD resistance assessment, differential phosphorylated proteins and SnRK1 kinase activities in transgenic plants will be analyzed，which will reveal the function of SnRK1β1 in response to Lxx infection in sugarcane and provide a new theory for molecular breeding of RSD resistance.