平菇谷氨酰胺合成酶（PoGS）同工酶基因的鉴定及功能分析

Pleurotus ostreatus is the most widely cultivated and consumed edible fungi in China, the yield and quality of which are significantly affected by the level of nitrogen supply. Thus, appropriate amount of nitrogen fertilizer was usually added to the cultivated medium to improve the yield of P. ostreatus. However, the molecular mechanisms of nitrogen uptake, transport and metabolism in P. ostreatus are still unclear. Numerous studies showed that as a key enzyme for nitrogen assimilation, glutamine synthetase is associated with many agronomic traits (including nitrogen-use, yield-related, quality-related traits, etc.) of crops. We have recently obtained three hypothetical glutamine synthetase coding genes (PoGS) in P. ostreatus by genomic analysis, and the coding sequence of each gene has been cloned. Based on our existing research, the functions of three PoGS genes and their roles in nitrogen assimilation will be characterized through enzymatic analysis, gene expression pattern analysis under different nitrogen levels and during different developing stages, gene knockout and overexpression analysis. This study is aimed to obtain the PoGS gene associated with nitrogen use efficiency, yield, and quality of P. ostreatus, thus providing theoretical basis and methodological guidance for cultivating high-efficiency, high-yield, and high-quality P. ostreatus varieties.

平菇是我国栽培量和消费量最大的食用菌，其产量和品质显著受到氮素供应水平的影响。为了提高平菇的产量，通常需要向培养料中添加适量的氮源，但平菇对氮素吸收、转运和代谢的分子机制尚不清楚。众多研究表明，谷氨酰胺合成酶（GS）作为氮素同化的关键酶，其活性与作物的氮素利用、产量性状和质量性状等农艺性状相关联性。申请人通过对平菇基因组分析获得了3个候选的谷氨酰胺合成酶基因（PoGS），并对其编码序列进行了克隆。本项目在已有研究基础上，拟通过酶学性质分析、不同施氮水平和不同生长发育阶段的基因表达模式分析、基因敲除及过表达分析等，对3个PoGS基因进行功能验证并分析其在氮素同化中的作用，旨在筛选出与平菇氮素利用率、产量和品质相关联的PoGS基因。从而为高效高产优质平菇新品种的培育提供重要的理论依据和方法指导。

平菇是我国主栽食用菌品种之一，2019年产量达到686.47万吨，位居我国食用菌第三位。我们前期研究发现平菇的产量和品质显著受到培养料含氮量和C/N比的影响，但是目前对平菇氮素吸收、转运、同化等的分子机制尚不清楚。谷氨酰胺合成酶（glutamine synthetase）是生物体氮素同化的关键酶，平菇基因组中存在三个功能注释为谷氨酰胺合成酶的假定基因（PoGS）。本项目首先分别对三个PoGS蛋白进行了原核表达、纯化，并测定了其谷氨酰胺合成酶活性和空间结构，明确了PoGS74和PoGS99蛋白具有谷氨酰胺合成酶活性，PoGS95蛋白不具有谷氨酰胺合成酶活性；PoGS74和PoGS99蛋白的酶学性质存在较大差异，PoGS74蛋白更耐热、PoGS99蛋白具有更高的铵根亲和性；自然状态下的PoGS74和PoGS99蛋白均为同源十聚体，有别于其它物种的GS空间结构。3个PoGS同工酶基因在不同发育时期和子实体不同部位的表达存在显著差异，PoGS74和PoGS95基因主要在营养生长阶段发挥作用，PoGS99基因主要在生殖生长阶段发挥作用；PoGS74、PoGS95和PoGS99基因分别在菌褶、菌褶、菌盖中的表达量最高。3个PoGS同工酶基因对氮源响应不同，短时间和长时间氮处理均可抑制PoGS99基因表达，短时间氮处理会抑制PoGS74、PoGS95基因表达，而长时间氮处理则会促进两个基因的表达。抑制谷氨酰胺合成酶活性会抑制平菇生长发育、改变氮代谢流。本研究明确了PoGS基因的功能，为平菇高产优质栽培提供了理论指导，为高效氮同化品种培育奠定了基础。