黑曲霉高效敲除体系构建及tpsA基因的敲除  

Construction of High Efficient Gene Knockout System in Aspergillus niger and Knockout of Trehalose-6-phosphate Synthase

作　　者：刘玲[1,2] 张鸿飞 张岚 秦郦[1,2] 王德培 LIU Ling;ZHANG Hong-fei;ZHANG Lan;QIN Li;WANG De-pei(State Key Laboratory of Food Nutrition and Safety,Tianjin 300457,China;Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education,Tianjin 300457,China;College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China)

机构地区：[1]工业发酵微生物教育部重点实验室,天津300457 [2]省部共建食品营养与安全国家重点实验室,天津300457 [3]天津科技大学生物工程学院,天津300457

出　　处：《食品研究与开发》2018年第22期124-130,共7页Food Research and Development

摘　　要：为完善高效的黑曲霉工程菌10142基因敲除体系,提高其基因敲除效率,构建含有致死基因单纯疱疹病毒胸苷激酶(herpes simplex virus thymidine kinase,HSVtk)的基因敲除质粒,通过农杆菌介导的黑曲霉转化(Agrobacterium tumefaciens-mediated transformation,ATMT)法,使致死基因随机插入黑曲霉基因组,其阳性转化子在添加10μmol/L的5-氟脱氧尿苷的培养基上无法生长,从而得到致死基因随机插入转化子的"反向筛选"方法。利用该方法对黑曲霉海藻糖-6-磷酸合成酶进行基因敲除,结果表明阳性转子占总转化子的比例为63%。成功构建高效黑曲霉基因敲除体系。An efficient method of gene knockout system was developed in Aspergillus niger 10142,an hyper-producer of citric acid fermentation.It constructed a binary vector containing lethal genes that a herpes simplex virus thymidine kinase(HSVtk)gene in T-DNA.By using Agrobacterium tumefaciens-mediated transformation(ATMT),the ectopic transformants did not grow on the medium addition of 10μmol/L 5-fluoro-2'-deoxyuridine.This method increased the proportion of positive transformants to select gene knockout transformants.Gene knockout experiments on trehalose-6-phosphate synthase(tpsA)in Aspergillus niger were performed using double-tagged vector containing lethal gene and hygromycin.The results showed that the frequency which refered to positive number account for total transformants was 63%.The successful construction of Aspergillus niger gene knockout system has provided an efficient technology platform for gene knockout experiments and researchment on the gene mechanisms of Aspergillus niger.

关 键 词：黑曲霉 基因敲除 致死基因 5-氟脱氧尿苷 海藻糖-6-磷酸合成酶 

分 类 号：Q933[生物学—微生物学]