三孢布拉霉遗传转化体系的构建及应用  

Construction and Application of Genetic Transformation System in Blakeslea trispora

作　　者：李娜[1] 曲音波[2] 杨培龙[3] 余晓斌[1] 罗玮[1] LI Na;QU Yinbo;YANG Peilong;YU Xiaobin;LUO Wei(School of Biotechnology,Jiangnan University,Wuxi 214122,China;State Key Laboratory of Microbial Technology,Shandong University,Qingdao 266237,China;Key Laboratory for Feed Biotechnology of the Ministry of Agriculture,Feed Research Institute Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]山东大学微生物技术国家重点实验室,山东青岛266237 [3]中国农业科学院饲料研究所农业农村部饲料生物技术重点实验室,北京100081

出　　处：《食品与生物技术学报》2021年第9期33-39,共7页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(21878123,21606105);中国博士后科学基金项目(2018M630525);微生物技术国家重点实验室开放课题项目(M2020-06);农业农村部饲料生物技术重点实验室开放课题项目(KLFB-FRI-202001)。

摘　　要：为解决三孢布拉霉转化过程中原生质体转化率低、孢子细胞壁厚难以导入外源载体等问题,本研究中构建了根癌农杆菌侵染原生质体的转化体系,同时克隆了与非同源末端连接修复途径相关的ku80基因,并将该转化体系应用于ku80的敲除中。将ku80基因敲除框插入双元载体pDHt-sk上构建了重组敲除载体pDH-85H3,通过化学转化法将敲除载体pDH-85H3导入根癌农杆菌LBA4404,并基于农杆菌介导法转化三孢布拉霉原生质体。结果表明,经潮霉素筛选和PCR鉴定,得到的20株转化子中,有18株的基因组插入了敲除框,转化率达90%。经鉴定有2株转化子发生了同源重组,敲除率为10%。该结论表明了根癌农杆菌介导三孢布拉霉原生质体转化技术的可行性。Blakeslea trispora(B.trispora),an important filamentous fungus,is used industrially in the mass production of carotenoids.The lack of a deep understanding of the genetic background of B.trispora and the shortage of genetic manipulation tools have hampered the genetic studies of B.trispora and the biological studies of carotenoid synthesis.For B.trispora,protoplast transformation rate is low and the spore cell wall is thick,which cause difficulty in introducing exogenous vectors into the cell.To solve this problem,this study constructed a transformation system mediated by Agrobacterium tumefaciens(A.tumefaciens)infecting protoplast,and cloned ku80 gene which was related with the non-homologous end joining repair pathways at the same time,and then applied the transformation system in the knockout of ku80.A recombinant knockout vector pDH-85H3 was constructed by inserting ku80 knockout frame into the binary vector pDHt-sk.And then the vector pDH-85H3 was introduced into A.tumefaciens LBA4404 by chemical transformation.Finally,the protoplast of B.trispora was transformed by A.tumefaciens-mediated method.By hygromycin screening and PCR analysis,there were 18 transformants in which the knockout box was inserted into the genome out of 20 transformants,and the transformation rate was 90%.Further analysis indicated that there were 2 transformants in which homologous recombination occurred and the knockout rate was 10%.The result indicated that A.tumefaciens mediated protoplast transformation in B.trispora was feasible.

关 键 词：三孢布拉霉 原生质体 KU80 根癌农杆菌 基因敲除 

分 类 号：Q939.97[生物学—微生物学]