内生真菌(Alternaria oxytropis)酵母氨酸还原酶基因功能互补株的构建及筛选鉴定  

Construction and Identification of sac Functional Complementary Strain in Alternaria oxytropis

作　　者：王钰 卢萍[1] 萨如拉 席领军 袁博 杜玲[1] Wang Yu;Lu Ping;Sarula;Xi Lingjun;Yuan Bo;Du Ling(College of Life Science and Technology,Inner Mongolia Normal University,Hohhot,010022)

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

出　　处：《分子植物育种》2020年第23期7777-7783,共7页Molecular Plant Breeding

基　　金：国家自然科学基金(31960130;31460235)资助。

摘　　要：本研究构建内生真菌酵母氨酸还原酶基因(saccharopine reductase,sac)功能互补载体pBARGPESac,制备sac缺失突变株M1的原生质体,将pBARGPE-Sac载体转化至M1原生质体后再生,构建和筛选出互补株C1。用RT-PCR技术检测转化子,并进行DNA测序,验证互补株。提取C1、M1及野生株OW7.8的苦马豆素(Swainsonine,SW),用HPLC-MS检测含量。结果显示:C1中检测出sac和hph的表达,sac cDNA测序正确。C1的SW水平高于OW7.8和M1(p<0.01),说明sac促进了该内生真菌中SW的合成。通过对sac功能的研究,为明晰真菌SW合成途径提供基础数据。The complementary vector(pBARGPE-Sac)of saccharopine reductase gene(sac)in Alternaria oxytropis was constructed.The protoplasts of sac knockout M1 mutant were extracted,and the pBARGPE-Sac was transformed into the protoplasts.Later the protoplasts were regenerated,and the complementary strain C1 was finally constructed.Transformants were detected by RT-PCR.Related DNA samples were sequenced and the complementary strains were verified.SW levels in C1,M1 and OW7.8 were performed by High Performance Liquid Chromatography-Mass Spectrometry(HPLC-MS).The results showed that sac and hph were expressed in C1.All sequences were proved true.The levels of SW in C1 were higher than that in OW7.8 and M1(p<0.01),which indicated sac promote the synthesis of SW.Through the study for the function of sac,the basic data was provided for understanding to SW synthesis pathway of this fungus.

关 键 词：内生真菌 酵母氨酸还原酶 互补株 

分 类 号：Q78[生物学—分子生物学]