内生真菌Alternaria oxytropis OW7.8中酵母氨酸还原酶基因启动子克隆表达及酶学性质  

Promoter Cloning and Expression,Enzymatic Properties of Saccharopine Reductase in Alternaria oxytropis OW7.8

作　　者：闵钰 赵利娜 卢萍[1] 杜玲[1] 袁博 姜凯 Min Yu;Zhao Lina;Lu Ping;Du Ling;Yuan Bo;Jiang Kai(College of Life Sciences and Technology,Inner Mongolia Normal University,Hohhot,O010022;Medical Laboratory College,Hebei North University,Zhangjiakou,075000)

机构地区：[1]内蒙古师范大学生命科学与技术学院,呼和浩特010022 [2]河北北方学院医学检验学院,张家口075000

出　　处：《分子植物育种》2023年第3期826-835,共10页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31960130,31460235)资助。

摘　　要：本研究利用TAIL-PCR法克隆小花棘豆内生真菌A.oxyrtropis OW7.8 sac启动子序列,并进行功能元件预测;构建pBARGPEI-mCherry-sac启动子表达载体,将该表达载体转化OW7.8原生质体,经再生培养得sac启动子转录激活功能株。提取分离A.oxyrtropis OW7.8中的Sac,对其进行酶活力、最适pH值、最适温度、K_(m)和V_(max)研究。结果表明:荧光显微镜下检测到转录激活功能株有明亮红色荧光,而野生株OW7.8无红色荧光,sac启动子驱动了红色荧光蛋白基因的转录。Sac逆向反应的最适pH值为7.0,最适温度为24℃;以酵母氨酸为底物的K_(m)=0.142 mmol/L、V_(max)=0.067μmol/min,以NADP+为底物的K_(m)=0.457 mmol/L、V_(max)=0.235μmol/min。为深入研究该内生真菌SW生物合成途径与调控机制提供了重要基础数据。The sac promoter of Saccharopine reductase(Sac)in endophytic fungus Alternaria oxytropis OW7.8 was cloned by TAIL-PCR,and its functional elements were predicted.The promoter expression vector pBARG-PEI-mCherry-sac was constructed and it was transformed into OW7.8 protoplasts.The sac promoter transcriptional activated strain was obtained through regeneration.Sac was isolated from A.oxyrtropis OW7.8,and enzyme activities,optimal pH,optimal temperature,K_(m)and V_(max)were determined.The results were as follows:The red fluorescence was detected under the fluorescence microscope in sac promoter transcriptional activated strain which showed the sac promoter drove the expression of RFP(Red fuorescent potein)gene.However,no red fluorescence was detected in OW7.8 strain.In reverse reaction,the optimal pH value of Sac was 7.0,and the optimal temperature was 24℃.The K_(m)=0.142 mmol/L,V_(max)=0.067μmol/min when saccharopine was selected as a substrate,while the K_(m)=0.457 mmol/L,V_(max)=0.235μmol/min when NADP+was selected as a substrate.These provide im portant basic data for further study of SW biosynthesis pathway and regulatory mechanism in A.oxyrtropis OW7.8.

关 键 词：内生真菌 苦马豆素 酵母氨酸还原酶 酶学性质 

分 类 号：Q78[生物学—分子生物学]