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.
作者
王钰
卢萍
萨如拉
席领军
袁博
杜玲
Wang Yu;Lu Ping;Sarula;Xi Lingjun;Yuan Bo;Du Ling(College of Life Science and Technology,Inner Mongolia Normal University,Hohhot,010022)
出处
《分子植物育种》
CAS
CSCD
北大核心
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。
关键词
内生真菌
酵母氨酸还原酶
互补株
Fungal endophyte(Alternaria oxytropis)
Saccharopine reductase
Complementary strain
