水稻OsUAP2基因生物信息学分析及原核表达活性鉴定  

Bioinformatics Analysis and Prokaryotic Expression Identification of OsUAP2 Gene in Rice

作　　者：肖桂青[1] 陈雅婷 周嘉豪[2] 文媛 高敏 张海文[2] 卢向阳[1] Xiao Guiqing;Chen Yating;Zhou Jiahao;Wen Yuan;Gao Min;Zhang Haiwen;Lu Xiangyang(College of Bioscience and Biotechnology,Hunan Agricultural University,Changsha,410128;The Biotechnology Research Institute(BRI)of Chinese Academy of Agricultural Science,Beijing,100081)

机构地区：[1]湖南农业大学生物科学技术学院,长沙410128 [2]中国农业科学院生物技术研究所,北京100081

出　　处：《分子植物育种》2022年第1期1-6,共6页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31701781);湖南省自然科学基金项目(2020JJ4365);湖南农业大学“1515”人才项目;湖南农业大学创新训练计划项目(s202010537077)共同资助。

摘　　要：UAGPase广泛存在于生物体,是糖代谢过程中一类重要酶。我们前期发现水稻Os UAP1具有单向催化UDPG分解代谢活性,Os UAP2与Os UAP1序列相似性极高。本研究对Os UAP2进行生物信息学分析,构建并表达重组蛋白,并鉴定其酶蛋白活性。结果表明,OsUAP2基因位于4号染色体,编码区序列长1482 bp,编码493个氨基酸;Os UAP2具有UDPGP保守结构域,无跨膜区域,N-端不含信号肽;二级结构分析显示,该蛋白含α-螺旋和无规则卷曲较多;同源性分析表明,Os UAP2与玉米UAGPase的亲缘关系最近。在16℃下经0.1 mmol/L IPTG诱导8 h,原核菌株BL21表达出分子量约55 kD可溶性重组蛋白。体外酶活性测定表明,Os UAP2能同时催化UDPG降解和合成,表现出双向可逆催化的特点。本研究结果为进一步深入研究OsUAP2基因的生物学功能提供了科学依据。UAGPase is very important enzyme in carbohydrate metabolism which exists widely in organisms.It was previously found that Os UAP1 can catalyze UDPG catabolism in one direction in rice.The sequences of Os UAP2 and Os UAP1 are highly similar.In this study,bioinformatics analysis on OsUAP2,prokaryotic expression and identification of its enzyme protein activity were conducted.The results showed that it was located on chromosome 4 with 1482 bp encoding 493 amino acids.There was a conserved domain named UDPGP in Os UAP2.There was no trans-membrane region and no N-terminal signal peptide.The secondary structure of Os UAP2 was mainlyα-helix and random coil.The phylogenetic tree indicated that it was highly conserved with UAGPase which was from maize.Recombinant protein construction,expression,and purification were performed.It showed that E.coli BL21 expressed soluble recombinant protein(HIS-OsUAP2)with a molecular weight of 55 kD,which was induced at 16℃and 0.1 mmol/L IPTG for 8 hours.Forward conversion of G-1-P to UDPG and reverse conversion of UDPG to G-1-P were observed with HIS-OsUAP2.That is to say,HIS-OsUAP2 could simultaneously catalyze the degradation and s ynthesis of UDPG,showing the characteristics of two-way reversible catalysis.These results provide scientific basis for further studying the biological function of OsUAP2.

关 键 词：水稻 OsUAP2 原核表达蛋白 酶活测定 

分 类 号：S511[农业科学—作物学]