江银辉,杨碧,吴昌学,周建奖
JIANG Yinhui,YANG Bi,WU Changxue,ZHOU Jianjiang

• 1:贵州医科大学地方病与少数民族疾病教育部重点实验室贵州省医学分子生物学重点实验室贵州医科大学分子生物学重点实验室

摘要(Abstract)：

目的:以吡啶硫胺抗性基因(ptrA)为筛选标记基因,建立黄曲菌遗传转化体系。方法:通过黄曲霉菌对潮霉素B(Hyg)、新霉素(G418)和嘧啶磺胺素(PT)的敏感性实验获得适合黄曲霉菌遗传转化筛选抗生素,用酶解的方法获得黄曲霉菌的原生质体;采用聚乙二醇(PEG)介导黄曲霉菌原生质体转化法,将携带ptrA的质粒pPTRI整合到黄曲菌株LD-F基因组DNA中,随机挑选7个转化子,通过聚合酶链反应(PCR)方法进行检测,验证ptrA是否整合到黄曲菌基因组DNA中。结果:0.2 mg/L PT可以完全抑制黄曲霉菌的菌丝生长,1%溶壁酶+1%纤维素酶+1%蜗牛酶处理黄曲霉菌丝2 h可得到大量的原生质体;PCR检测结果显示,质粒pPTRI能够成功整合到黄曲菌基因组。结论:以PT为抗性筛选,建立PEG介导黄曲霉菌的遗传转化体系。
Objective: To establish the genetic transformation system of Aspergillus flavus by using pyridine thiamine resistance gene(ptrA) as screening marker gene. Methods: Through the sensitivity test of Aspergillus flavus to hygromycin B(Hyg), neomycin(G418) and pyrimidine sulfonamidine(PT), the protoplasts of Aspergillus flavus were obtained by genetic transformation and screening of antibiotics, and the protoplasts of Aspergillus flavus were obtained by enzyme hydrolysis. The plasmid pPTRI carrying ptrA was integrated into the genomic DNA of Aspergillus flavus LD-F by PEG mediated protoplast transformation. Seven transformants were randomly selected and detected by PCR to verify whether ptrA was integrated into the genomic DNA of Aspergillus flavus. Results: 0.2 mg/L PT completely inhibited the hyphal growth of Aspergillus flavus. A large amount of protoplasts were obtained by 1% lysing enzyme+1% cellulose+1% snail enzyme treatment for 2 h of Aspergillus flavus. The results of PCR showed that plasmid pPTRI could be successfully integrated into the genome of Aspergillus flavus. Conclusion: The genetic transformation system of Aspergillus flavus mediated by PEG is established by using PT as resistance screening.

关键词(KeyWords)： 黄曲霉菌;吡啶磺胺素;PEG介导;原生质体;遗传转化
aspergillus flavus;taurosulfone;PEG-mediate;protoplast;genetic transformation

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(31660012);; 贵州省科学计划项目[黔科合基础(2019)1277];; 贵州省科技合作计划项目[黔科合LH字(2016)7348];; 贵阳市科技计划项目[筑科合同(20161001)032];; 贵州省教育厅青年科技人才成长项目[黔教合KY字(2016)146];; 贵州省科技计划项目[黔科合平台人才(2017)5652]

作者(Author): 江银辉,杨碧,吴昌学,周建奖
JIANG Yinhui,YANG Bi,WU Changxue,ZHOU Jianjiang

DOI: 10.19367/j.cnki.1000-2707.2019.12.005

参考文献(References)：

• [1] SABIR N,IKRAM A,GARDEZI A,et al.Native valve dual pathogen endocarditis caused by Burkholderia cepacia and Aspergillus flavus-a case report[J].Jmmol Case Reports,2018,5(9):1-4
• [2] HADRICH I,DRIRA I,NEJI S,et al.Microsatellite typing of Aspergillus flavus from clinical and environmental avian isolates[J].Journal of Medical Microbiology,2013,62:121-125.
• [3] WINDHAM G L,WILLIAMS W P,MYLROIE E,et al.A histological study of Aspergillus flavus colonization of wound inoculated maize kernels of resistant and susceptible maize hybrids in the field[J].Frontiers in Microbiology,2018,9:799.
• [4] BHATNAGARMATHUR P,SUNKARA S,BHATNAGARPANWAR M,et al.Biotechnological advances for combating Aspergillus flavus and aflatoxin contamination in crops[J].Plant Science,2015,234:119-132.
• [5] CASQUETE R,BENITO M J,DEGULACORDOBA M,et al.The growth and aflatoxin production of Aspergillus flavus strains on a cheese model system are influenced by physicochemical factors[J].Journal of Dairy Science,2017,100(9):6987-6996.
• [6] CARY J W,GILBERT M K,LEBAR M D,et al.Aspergillus flavus secondary metabolites:More than just aflatoxins[J].Food Safety,2018,6(1):7-32.
• [7] SEYEDMOUSAVI S,MOUTON J W,MELCHERS W J G,et al.The role of azoles in the management of azole-resistant aspergillosis:from the bench to the bedside[J].Drug Resistance Updates,2014,17(3):37-50.
• [8] NATESAN S K,LAMICHCHANE A K,SSWAMINATHAN S,et al.Differential expression of ATP-binding cassette and/or major facilitator superfamily class efflux pumps contributes to voriconazole resistance in Aspergillus flavus[J].Diagnostic Microbiology and Infectious Disease,2013,76(4):458-463.
• [9] PAUL R A,RUDRAMURTHY S M,MEIS J F,et al.A novel Y319H substitution in CYP51C associated with azole resistance in Aspergillus flavus[J].Antimicrobial Agents and Chemotherapy,2015,59(10):6615-6619.
• [10]LOHAR J M,HARRIS-COWARD P Y,CARY J W,et al.rtfA,a putative RNA-Pol II transcription elongation factor gene,is necessary for normal morphological and chemical development in Aspergillus flavus[J].Applied Microbiology and Biotechnology,2016,100(11):5029-5041.
• [11]FORSETH R R,AMAIKE S,SCHWENK D,et al.Homologous NRPS-like gene clusters mediate redundant small-molecule biosynthesis in Aspergillus flavus[J].Angewandte Chemie International Edition,2013,52(5):1590-1594.
• [12]BHATNAGAR-MATHUR P,SUNKARA S,BHATNAGAR-PANWAR M,et al.Biotechnological advances for combating Aspergillus flavus and aflatoxin contamination in crops[J].Plant Science,2015,234:119-132.
• [13]SUN Y,NIU Y,HE B,et al.A dual selection marker transformation system using Agrobacterium tumefaciens for the industrial Aspergillus oryzae 3.042[J].Journal of Microbiology and Biotechnology,2019,29(2):230-234.
• [14]KUBODERA T,YAMASHITA N,NISHIMURA A.Pyrithiamine resistance gene (ptrA) of Aspergillus oryzae:cloning,characterization and application as a dominant selectable marker for transformation[J].Bioscience,Biotechnology and Biochemistry,2000,64(7):1416-1421.
• [15]SUN Y,NIU Y,HE B,et al.A dual selection marker transformation system using Agrobacterium tumefaciens for the industrial Aspergillus oryzae 3.042[J].Journal of Microbiology and Biotechnology,2019,29(2):230-234.
• [16]WOLOSHUK C P ,SEIP E R ,PAYNE G A ,et al.Genetic transformation system for the aflatoxin-producing fungus Aspergillus flavus[J].Applied and Environmental Microbiology,1989,55(1):86-90.
• [17]MICHIELSE C B,HOOYKAAS P J J,VAN DEN HONDEL C A,et al.Agrobacterium-mediated transformation as a tool for functional genomics in fungi[J].Current Genetics,2005,48(1):1-17.
• [18]HAN G,SHAO Q,LI C,et al.An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus[J].Journal of Microbiology,2018,56(5):356-364.
• [19]ADELEYE T,KAREEM S,DAIRO O,et al.Studies on amylase from protoplast fusants of Aspergillus species using response surface methodology[J].Access Microbiology,2019,1(1A):1.
• [20]储韬,黄国玉,赵志伟,等.黄曲霉菌原生质体的制备和再生技术优化[J].激光生物学报,2014,23(4):380-384.

文章评论(Comment)：