食品与发酵工业 >

2022 , Vol. 48 >Issue 2: 15 - 19

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.027893

研究报告

黑曲霉pyrG遗传转化系统的构建及应用

  • 胡江峰 ,
  • 刘舒雯 ,
  • 杨焱 ,
  • 李林霖 ,
  • 李迎凯 ,
  • 张健
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  • 1(工业发酵微生物教育部重点实验室,天津科技大学 生物工程学院,天津,300457)
    2(天津市经济贸易学校,天津,300381)
硕士研究生(张健副研究员为通信作者,E-mail:zj96sk@tust.edu.cn)

收稿日期: 2021-04-28

  修回日期: 2021-05-15

  网络出版日期: 2022-02-28

基金资助

国家自然科学基金面上项目(31471725);山东省重点研发计划(领军人才培育项目)(2016GRC3201)

收起

Construction and application of Aspergillus niger pyrG genetic transformation system

  • HU Jiangfeng ,
  • LIU Shuwen ,
  • YANG Yan ,
  • LI Linlin ,
  • LI Yingkai ,
  • ZHANG Jian
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  • 1(Key Laboratory of Industrial Fermentation Microbiology Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China)
    2(Tianjin Economics and Trade School, Tianjin 300381, China)

Received date: 2021-04-28

  Revised date: 2021-05-15

  Online published: 2022-02-28

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摘要

用于黑曲霉遗传操作的筛选标记基因非常有限,而基因改造往往涉及诸多基因的敲除或表达,有限的筛选标记基因难以满足需要。该文构建了以pyrG为筛选标记的黑曲霉遗传转化系统,并利用该系统使红色荧光蛋白rfp在黑曲霉中成功表达。首先,运用同源重组原理完全敲除pyrG基因,在含有5-氟乳清酸和尿苷/尿嘧啶的培养基平板进行筛选,获得1株稳定遗传的尿苷/尿嘧啶营养缺陷型菌株黑曲霉ng-1。然后构建了以pyrG作为回补标记,以三磷酸甘油醛脱氢酶启动子gpdA来调节rfp基因表达的质粒,将质粒转入农杆菌AGL1并侵染黑曲霉菌株ng-1,成功获得了黑曲霉rfp表达菌株,表明pyrG筛选标记可成功用于黑曲霉的遗传转化。

关键词： 黑曲霉; pyrG; 根癌农杆菌介导的转化法; 筛选标记; 红色荧光蛋白

本文引用格式

胡江峰 , 刘舒雯 , 杨焱 , 李林霖 , 李迎凯 , 张健 . 黑曲霉pyrG遗传转化系统的构建及应用[J]. 食品与发酵工业, 2022 , 48(2) : 15 -19 . DOI: 10.13995/j.cnki.11-1802/ts.027893

Abstract

Few selective marker genes are used in the genetic operation of Aspergillus niger. However, genetic modification often involves the knockout or expression of many genes, and the limited selective marker genes are difficult to meet the needs. In this study, genetic transformation system was constructed using pyrG as selective marker in A. niger and the red fluorescent protein rfp in A. niger was expressed using this system. First, the pyrG gene was knocked out by targeted gene replacement using Agrobacterium tumefaciens mediated transformation. The stable genetic uridine/uracil auxotrophic strain A. niger ng-1 was obtained on medium containing 5-fluoroorotic acid and uridine/uracil. Then we constructed the rfp gene-expression plasmid which contained the rfp gene driven by the glyceraldehyde triphosphate dehydrogenase promoter gpdA and the selective marker pyrG. The plasmid was transferred to A. tumefaciens AGL1 and infected the A. niger ng-1, so as to obtain rfp gene-expression A. niger strains. This work showed that the genetic transformation system using the pyrG as a selective marker is effective and feasible in A. niger.

Key words： Aspergillus niger; pyrG; Agrobacterium tumefaciens-mediated transformation; selective marker; red fluorescent protein

参考文献

[1] LI C, ZHOU J W, DU G C, et al.Developing Aspergillus niger as a cell factory for food enzyme production[J].Biotechnology Advances, 2020, 44:107630.
[2] 谢承佳, 庄琪.酱油制曲研究进展[J].中国调味品, 2017, 42(6):77-80.
XIE C J, ZHUANG Q.Research progress of koji making for fermented soy sauce[J].China Condiment, 2017, 42(6):77-80.
[3] 李莉. 用黑曲霉、绿色木霉制备酱油种曲的工艺参数确定[J].中国调味品, 2008, 33(9):42-44;52.
LI L.Ascertainment of optimum technology parameters for producting seed koji using Aspergillus niger and Trichoderma viride[J].China Condiment, 2008, 33(9):42-44;52.
[4] 孙士健, 王丽娟, 秦郦, 等.复合诱变筛选高产柠檬酸黑曲霉及其发酵研究[J].食品研究与开发, 2019, 49(17):194-200.
SUN S J, WANG L J, QIN L, et al.Compound mutation and screening Aspergillus niger with high yield of citric acid and its fermentation research[J].Food Research and Development, 2019, 49(17):194-200.
[5] 孙菁, 任文琪, 许勤虎, 等.以amdS为筛选标记的黑曲霉遗传转化体系的建立[J].中国农业科技导报, 2020, 22(9):179-187.
SUN J, REN W Q, XU Q H, et al.Transformation system of Aspergillus niger using amdS as a dominant selective marker[J].Journal of Agricultural Science and Technology, 2020, 22(9):179-187.
[6] ORTEGA-ESCALANTE J A, KWOK O, MILLER S M.New selectable markers for Volvox carteri transformation[J].Protist, 2019, 170(1):52-63.
[7] LIU J J, XIE Z P, SHIN H D, et al.Rewiring the reductive tricarboxylic acid pathway and L-malate transport pathway of Aspergillus oryzae for overproduction of L-malate[J].Journal of Biotechnology, 2017, 253:1-9.
[8] SUZUKI S, TADA S, FUKUOKA M, et al.A novel transformation system using a bleomycin resistance marker with chemosensitizers for Aspergillus oryzae[J].Biochemical and Biophysical Research Communications, 2009, 383(1):42-47.
[9] 王伯华. 橙色红曲菌中基于pyrG标记的同源转化系统的建立[D].南昌:南昌大学, 2010.
WANG B H.Development of a homologous transformation system for Monascus aurantiacus based on the pyrG gene[D].Nanchang:Nanchang University, 2010.
[10] HARTINGSVELDT W, MATTERN I E, ZEIJL C M J, et al.Development of a homologous transformation system for Aspergillus niger based on the pyrG gene[J].Molecular and General Genetics, 1987, 206(1):71-75.
[11] NGUYEN K T, HO Q N, PHAM T H, et al.The construction and use of versatile binary vectors carrying pyrG auxotrophic marker and fluorescent reporter genes for Agrobacterium-mediated transformation of Aspergillus oryzae[J].World Journal of Microbiology and Biotechnology, 2016, 32(12):1-9.
[12] UNKLES S E, CAMPBELL E I, DE RUITER-JACOBS Y M J T, et al.The development of a homologous transformation system for Aspergillus oryzae based on the nitrate assimilation pathway:A convenient and general selection system for filamentous fungal transformation[J].Molecular and General Genetics, 1989, 218(1):99-104.
[13] 李方方, 潘力, 曾沛斌.酱油工业生产菌沪酿3042基因工程转化体系的构建[J].食品工业科技, 2009,13(6):94-95;99.
LI F F, PAN L, ZENG P B.Construction of genetic engineering transforming system of Aspergillus oryzae HuNiang 3042 used in soy sauce industry[J].Science and Technology of Food Industry, 2009,13(6):94-95;99.
[14] THAI H D, NGUYEN B P T, NGUYEN V M, et al.Development of a new Agrobacterium-mediated transformation system based on a dual auxotrophic approach in the filamentous fungus Aspergillus oryzae[J].World Journal of Microbiology and Biotechnology, 2021, 37(6):92.
[15] BOEKE J D, TRUEHEART J, NATSOULIS G, et al.5-Fluoroorotic acid as a selective agent in yeast molecular genetics[J].Methods in Enzymology, 1987, 154:164-175.
[16] 陈英, 王培娟, 张文君, 等.mCherry红色荧光标记乳酸菌的融合表达系统构建及应用[J].生物工程学报, 2019, 35(3):492-504.
CHEN Y, WANG P J, ZHANG W J, et al.Construction and application of mCherry red fluorescent protein fusion expression system in lactic acid bacteria[J].Chinese Journal of Biotechnology, 2019, 35(3):492-504.
[17] ZHAO P, YE M H, WANG R Q, et al.Systematic identification and functional analysis of potato (Solanum tuberosum L.) bZIP transcription factors and overexpression of potato bZIP transcription factor StbZIP-65 enhances salt tolerance[J].International Journal of Biological Macromolecules, 2020, 161:155-167.
[18] 陈阳霞, 顾桉菁, 梁宇恒, 等.电穿孔介导马尔尼菲青霉转化体系的建立和优化[J].中国真菌学杂志, 2014, 9(1):12-15.
CHEN Y X, GU A J, LIANG Y H, et al.Electroporation-mediate transformation of Penicillium marneffei and optimization of the transforming conditions[J].Chinese Journal of Mycology, 2014, 9(1):12-15.
[19] 李载平. 分子克隆实验指南(第3版)[J].科学通报, 2002, 47(24):1 888.
LI Z P.Molecular cloning experiment guide(the 3rd edition)[J].Chinese Science Bulletin, 2002, 47(24):1 888.
[20] 黎明, 刘萌, 黄云雁, 等.根癌农杆菌介导的黑曲霉遗传转化体系的建立及优化[J].中国生物工程杂志, 2012, 32(1):56-63.
LI M, LIU M, HUANG Y Y, et al.Establishment and optimization of Agrobacterium tumefaciens mediated transformation system of Aspergillus niger[J].China Biotechnology, 2012, 32(1):56-63.
[21] 毕付提, 史亚楠, 张久祎, 等.米曲霉3.042尿苷/尿嘧啶营养缺陷型遗传转化体系的构建[J].食品研究与开发, 2021, 42(3):189-195.
BI F T, SHI Y N, ZHANG J Y, et al.Construction of Aspergillus oryzae 3.042 uridine/uracil auxotrophy genetic transformation system[J].Food Research and Development, 2021, 42(3):189-195.
[22] 裴静娴, 王月刚, 张艺军, 等.p53RFP基因启动子荧光素酶报告基因载体的构建及鉴定[J].广东医学, 2017, 38(2):170-172.
PEI J X, WANG Y G, ZHANG Y J, et al.Construction and identification of luciferase reporter plasmid containing p53RFP gene promoter[J].Guangdong Medical Journal, 2017, 38(2):170-172.
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