糙皮侧耳谷氨酰胺合成酶PoGlnA的鉴定及氮源响应分析  

Identification and nitrogen response of glutamine synthetase PoGlnA from Pleurotus ostreatus

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作  者:魏志霞 刘元栋[1,2] 李佳涛 胡延如 申进文 戚元成[1] 王风芹[1] 文晴[1] WEI Zhixia;LIU Yuandong;LI Jiatao;HU Yanru;SHEN Jinwen;QI Yuancheng;WANG Fengqin;WEN Qing(College of Life Sciences,Henan Agricultural University,Zhengzhou 450002,Henan,China;Zhoukou Academy of Agricultural Sciences,Zhoukou 466000,Henan,China)

机构地区:[1]河南农业大学生命科学学院,河南郑州450002 [2]周口市农业科学院,河南周口466000

出  处:《菌物学报》2025年第2期68-80,共13页Mycosystema

基  金:国家自然科学基金(31701980);国家现代农业技术体系建设专项(CARS-20);河南省科技攻关项目(232102111111);河南省农业良种联合攻关项目(2022030101)。

摘  要:谷氨酰胺合成酶是氮素同化的关键酶,在生物体氮代谢过程中发挥重要作用。本文从糙皮侧耳中克隆获得了一个GlnA家族的谷氨酰胺合成酶基因PoGlnA,首先,对PoGlnA蛋白进行了异源表达、纯化和酶活性测定,结果表明:PoGlnA基因的编码区长1446 bp,编码481个aa;PoGlnA蛋白的分子量为53 kDa,具有谷氨酰胺合成酶活性。然后,将PoGlnA蛋白与其他已报道的谷氨酰胺合成酶蛋白进行了系统发育关系分析,结果显示:PoGlnA蛋白与细菌来源的GlnA蛋白具有相对更近的亲缘关系,与已报道的食用菌来源的谷氨酰胺合成酶亲缘关系较远。最后,从转录水平和翻译水平分析了PoGlnA基因对氮饥饿和不同种类氮源的响应,结果显示:氮饥饿、天冬氨酸、天冬酰胺、亮氨酸和谷氨酸处理可促进PoGlnA基因表达,铵态氮和谷氨酰胺处理对其表达无影响,但硝态氮处理抑制其表达。此外,本文还通过抗原表位分析获取了PoGlnA蛋白的优势抗原肽段,并制备了特异性的PoGlnA多克隆抗体,为深入揭示PoGlnA基因的生物学功能奠定了基础。Glutamine synthetase is the key enzyme of nitrogen assimilation and plays a critical role in nitrogen metabolism.In this study,the glutamine synthetase coding gene PoGlnA belonging to the GlnA family was cloned from Pleurotus ostreatus.The PoGlnA protein was heterologously expressed and purified and the glutamine synthetase activity was determined.The results showed that the CDS sequence of PoGlnA gene was 1446 bp,coding 481 aa.The molecular weight of PoGlnA protein was 53 kDa,possessing the glutamine synthetase activity.The phylogenetic relationship between PoGlnA protein and other reported glutamine synthetase proteins was analyzed.The result showed that there was a relatively close relationship between PoGlnA and GlnA protein from bacteria,but the relationships between PoGlnA protein and the reported glutamine synthetase protein from edible fungi were distant.The responses of PoGlnA gene to nitrogen starvation and different nitrogen sources were analyzed at the transcriptional and the translational level.The results showed that nitrogen starvation,and Asp,Asn,Leu,Glu treatment could promote PoGlnA gene expression,while ammonium and Gln treatment had no significant influence on the expression,but nitrate treatment inhibited the expression.In addition,the peptide containing dominant epitopes of PoGlnA was obtained by antigen epitope analysis,and the specific anti-PoGlnA PcAb was furtherly prepared,laying a foundation for elucidating the physiological function of PoGlnA gene.

关 键 词:糙皮侧耳 谷氨酰胺合成酶 原核表达 酶活性 氮源响应 

分 类 号:S646.14[农业科学—蔬菜学]

 

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