禾谷镰刀菌FgGDH基因的克隆和酶促动力学分析  

Cloning and Enzyme Kinetics Analysis of FgGDH Gene from Fusarium graminearum

作　　者：罗琼 何勇 燕璐 曾慧 夏清珊 王艳 林建中[1] 刘选明[1] LUO Qiong;HE Yong;YAN Lu;ZENG Hui;XIAQing-Shan;WANG Yan;LIN Jian-Zhong;LIU Xuan-Ming(College of Biology/Hunan Province Key Laboratory of Plant Functional Genomics and Developmental Regulation,Hunan University,Changsha 410082,China)

机构地区：[1]湖南大学生物学院/植物功能基因组学和发育调控湖南省重点实验室,长沙410082

出　　处：《农业生物技术学报》2021年第8期1594-1603,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31571635;31871595);湖南省科技重大专项(2018NK1010);湖南省自然科学基金(2020JJ4004);中国博士后科学基金(2020M682561);广西水稻遗传育种重点实验室开放课题(2018-05-Z06-KF05);杂交水稻国家重点实验室(湖南杂交水稻研究中心)开放课题(2019KF02)。

摘　　要：谷氨酸脱氢酶(glutamate dehydrogenase,GDH)是广泛存在于生物体中与氮代谢相关的酶,而低等生物的GDH基因常被用于改良作物的氮同化效率。为了发掘优质GDH基因用于作物的遗传改良,本研究从禾谷镰刀菌(Fusarium graminearum)中克隆了FgGDH基因,然后原核表达和纯化FgGDH,并在体外酶促反应体系中测定其酶促动力学特性。结果显示,FgGDH催化氨化反应的酶活性显著大于去氨化反应的酶活性,说明FgGDH更倾向于催化NH_(4)^(+)和α-酮戊二酸(α-ketoglutaric acid,2-OG)合成谷氨酸,促进NH_(4)^(+)的同化。此外,FgGDH对NH_(4)^(+)和2-OG的Km值分别为(3.71±0.21)mmol/L和(6.44±0.32)mmol/L,均显著低于水稻OsGDH4的相应Km值,说明FgGDH对NH_(4)^(+)和2-OG的亲和力显著高于水稻内源GDH,具有较高的氨同化效率。本研究为后续利用FgGDH基因用于水稻等作物氮同化效率的遗传改良提供了理论基础。Glutamate dehydrogenase(GDH)is an enzyme widely found in organisms related to nitrogen metabolism,and GDH genes of lower organisms are often used for the improvement of nitrogen assimilation efficiency in crops.In order to explore the high quality GDH genes for genetic improvement of crops,FgGDH gene was cloned from Fusarium graminearum,the protein FgGDH was prokaryotic expressed and purified,and its enzymatic kinetics characteristics were determined in vitro in this study.The results showed that the aminating activity of FgGDH was significantly higher than that of deaminating activity,indicating that FgGDH is more inclined to catalyze NH_(4)^(+)andα-ketoglutaric acid(2-OG)to generate glutamic acid and promotes the assimilation of NH_(4)^(+).In addition,the Km values of FgGDH for NH_(4)^(+)and 2-OG were(3.71±0.21)mmol/L and(6.44±0.32)mmol/L,respectively,which were significantly lower than those of OsGDH4 in rice.These results indicate that the affinity of FgGDH for NH_(4)^(+)and 2-OG is marked higher than those of endogenous OsGDH4 in rice,and FgGDH has a higher ammonia assimilation efficiency.This study provides a theoretical basis for the genetic improvement of nitrogen assimilation efficiency in rice and other crops by using FgGDH gene.

关 键 词：谷氨酸脱氢酶(GDH) 原核表达 酶促动力学 氮同化 

分 类 号：S182[农业科学—农业基础科学]