WANG Juan,GONG Yanfen,LAI Yapeng,et al.The effect of RNA interference of MHR1 on cellulase activity in Myceliophthora thermophila[J].Journal of Shenzhen University Science and Engineering,2018,35(No.1(001-110)):15-21.[doi:10.3724/SP.J.1249.2018.01015]





The effect of RNA interference of MHR1 on cellulase activity in Myceliophthora thermophila
1)深圳大学生命与海洋科学学院,深圳市微生物基因工程重点实验室,广东深圳 518060
2)深圳市海洋生物资源与生态环境重点实验室,广东深圳 518060
WANG Juan1 GONG Yanfen12 LAI Yapeng1 DENG Tingting1 XIE Ning1 and LIU Gang1
1) College of Life Sciences and Oceanography, Shenzhen Key Laboratory of Microbiology and Gene Engineering, Shenzhen University, Shenzhen 518060, Guangdong Province, P.R.China
2) Shenzhen Key Laboratory of Marine Bioresources and Ecology, Shenzhen 518060, Guangdong Province, P.R.China
thermophilic fungi Myceliophthora thermophila RNA interference cellulase MHR1 factor cellulose biomass
Q 939.9
利用RNA-Seq技术,筛选到潜在的负调控因子 MHR1(fungal_TF_MHR1). 为研究MHR1与纤维素酶基因表达之间的关联,设计基因mhr1的RNA干扰序列,并使用丙酮酸脱羧酶强启动子pdc成功构建mhr1基因的RNA干扰表达载体,经原生质体转化,获得一株干扰效率较高的阳性转化子MtR5.通过RT-qPCR、酶活测定和胞外蛋白浓度测定等分析发现,在诱导和非诱导培养条件下,转化子MtR5的胞外蛋白浓度、纤维素酶活性以及主要纤维素酶基因的表达量均高于野生型的.干扰转录因子MHR1可增加纤维素酶基因的表达量,研究表明,MHR1是一种与纤维素酶基因表达调控相关的转录因子.
The potential negative regulatory factor MHR1 was screened by RNA-Seq technique. A positive transformant MtR5 was successfully obtained by constructing the RNAi expression vector for mhr1 through the designing of the RNA interference sequence aimed at mhr1 and the use of the strong promoter of pdc gene. Real-time quantitative PCR, enzymatic and protein concentration determination show that the cellulase activities, extracellular protein concentration, and main cellulase genes expression of the transformant MtR5 are higher than those of the wild type under both inducing and non-inducing conditions. The above results elucidate the relationship between MHR1 and cellulase gene regulation for the first time and the association between the protein MHR1 and carbon catabolite repression. MHR1 works as a novel transcription factor associated with the cellulase gene expression and it is a repressor in carbon catabolite repression.


[1] CHUNDAWAT S S, BECKHAM G T, HIMMEL M E,et al. Deconstruction of lignocellulosic biomass to fuels and chemicals[J]. Annual Review of Chemical and Biomolecular Engineering, 2011, 2(2): 121-145.
[2] JUTURU V, WU Jinchuan. Microbial cellulases: engineering, production and applications[J].Renewable and Sustainable Energy Reviews, 2014, 33: 188-203.
[3] 来亚鹏,邓婷婷,刘 刚,等. 同源过表达BglR对嗜热毁丝霉β-葡萄糖苷酶活性的影响[J]. 中国生物工程杂志,2017,37(7):64-71.
LAI Yapeng, DENG Tingting, LIU Gang, et al. The influence of homologous overexpression of BglR on β-glucosidase activities in Myceliophthora thermophila[J]. China Biotechnology, 2017,37(7):64-71.(in Chinese)
[4] MOUCHACCA J. Thermophilic fungi: biodiversity and taxonomic status[J]. Cryptogamie Mycologie,1997,18(1):19-69.
[5] MAHESHWARI R, BHARADWAJ G, BHAT M K. Thermophilic fungi: their physiology and enzymes[J].Microbiology and Molecular Biology Reviews,2000,64(3):461-488.
[6] MORGENSTERN I, POWLOWSKI J, ISHMAEL N,et al. A molecular phylogeny of thermophilic fungi[J].Fungal Biology,2012,116(4):489-502.
[7] BERKA R M, GRIGORIEV I V, OTILLAR R,et al. Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris[J].Nature Biotechnology,2011,29(10):922-927.
[8] MATSAKAS L, ANTONOPOULOU I, CHRISTAKOPOULOS P. Evaluation of Myceliopthora thermophila as an enzyme factory for the production of thermophilic cellulolytic enzymes[J]. BioResources, 2015, 10(3):5140-5158.
[9] VISSER H, JOOSTEN V, PUNT P J,et al. Research: Development of a mature fungal technology and production platform for industrial enzymes based on a Myceliophthora thermophila isolate, previously known as Chrysosporium lucknowense C1[J]. Industrial Biotechnology, 2011, 7(3): 214-223.
[10] KOLBUSZ M A, DI FALCO M, ISHMAEL N,et al. Transcriptome and exoproteome analysis of utilization of plant-derived biomass by Myceliophthora thermophila [J]. Fungal Genetics and Biology, 2014, 72: 10-20.
[11] SINGH B. Myceliophthora thermophila syn. Sporotrichum thermophile: a thermophilic mould of biotechnological potential[J]. Critical Reviews in Biotechnology, 2016, 36(1): 59-69.
[12] LIU Qian, GAO Ranran, LI Jingen,et al. Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering[J].Biotechnology for Biofuels,2017,10(1):1.
[13] WANG Juan, WU Yaning, GONG Yanfen,et al. Enhancing xylanase production in the thermophilic fungus Myceliophthora thermophila by homologous overexpression of Mtxyr1[J].Journal of Industrial Microbiology & Biotechnology,2015,42(9):1233-1241.
[14] KLAUBAUF S, NARANG H M, POST H,et al. Similar is not the same: differences in the function of the (hemi-) cellulolytic regulator XlnR (Xlr1/Xyr1) in filamentous fungi[J]. Fungal Genetics and Biology, 2014, 72: 73-81.
[15] YANG Fan, GONG Yanfen, LIU Gang,et al. Enhancing cellulase production in thermophilic fungus myceliophthora thermophila ATCC42464 by RNA interference of cre1 gene expression[J]. Journal of Microbiology and Biotechnology, 2015, 25(7):1101-1107.
[16] 杨帆. 转录因子CRE1及ACE1在嗜热毁丝霉纤维素酶基因表达调控中的作用[D]. 深圳:深圳大学,2015.
YANG Fan. The role of transcription factors CRE1 and ACE1 in regulation of cellulase gene expression of Myceliophthora thermophila ATCC42464 [D]. Shenzhen: Shenzhen University, 2015.(in Chinese)
[17] AMORE A, GIACOBBE S, FARACO V. Regulation of cellulase and hemicellulase gene expression in fungi[J]. Current genomics, 2013, 14(4): 230-249.
[18] PORTNOY T, MARGEOT A, SEIDL-SEIBOTH V, et al. Differential regulation of the cellulase transcription factors XYR1, ACE2, and ACE1 in Trichoderma reesei strains producing high and low levels of cellulose[J]. Eukaryotic cell, 2011, 10(2): 262-271.
[19] MORIN R, BAINBRIDGE M, FEJES A,et al. Profiling the HeLa S3 transcriptome using randomly primed cDNA and massively parallel short-read sequencing[J].BioTechniques,2008,45(1):81-94.
[20] CHU Yongjun, COREY D R. RNA sequencing: platform selection, experimental design, and data interpretation[J].Nucleic Acid Therapeutics,2012,22(4):271-274.
[21] WANG Zhong, GERSTEIN M, SNYDER M. RNA-Seq: a revolutionary tool for transcriptomics[J].Nature Reviews Genetics,2009,10(1):57-63.
[22] TRAPNELL C, WILLIAMS B A, PERTEA G,et al. Transcript assembly and abundance estimation from RNA-Seq reveals thousands of new transcripts and switching among isoforms[J]. Nature biotechnology, 2010, 28(5): 511-515.
[23] SONESON C, DELORENZI M. A comparison of methods for differential expression analysis of RNA-seq data[J]. BMC bioinformatics, 2013, 14(1): 91.
[24] PENTTIL M, NEVALAINEN H, RTT M,et al. A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei[J].Gene,1987,61(2):155-164.
[25] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method[J].Methods,2001,25(4):402-408.
[26] EVELEIGH D E, MANDELS M, ANDREOTTI R, et al. Measurement of saccharifying cellulase[J].Biotechnology for Biofuels,2009,2(1):21.
[27] MANSOUR A A, DA COSTA A, ARNAUD T, et al. Review of lignocellulolytic enzyme activity analyses and scale-down to microplate-based assays[J]. Talanta, 2016, 150: 629-637.
[28] 麦国琴,许晓萍,余翠媚,等.产木聚糖酶和纤维素酶真菌的酶学性质分析[J]. 食品研究与开发,2011, 32(9) :179-183.
MAI Guoqin, XU Xiaoping, YU Cuimei, et al. Screening of cellulase and xylanase-pruducing fungi from mangrove soil and characterization of the enzymatic reactions[J]. Food Research and Development, 2011, 32(9) :179-183.(in Chinese)
[29] BAILEY M J, BIELY P, POUTANEN K. Interlaboratory testing of methods for assay of xylanase activity[J]. Journal of Biotechnology, 1992, 23(3): 257-270.
[30] 周娇娇,佘炜怡,王浩入,等. 5-氮杂-2-脱氧胞苷对里氏木霉产纤维素酶的影响[J]. 深圳大学学报理工版,2017,34(2):122-131.
ZHOU Jiaojiao, SHE Huiyi, WANG Haoru, et al. Effect of 5-Aza-2′-deoxycytidine on the expression of cellulases in Trichoderma reesei[J].Journal of Shenzhen University Science and Engineering,2017,34(2):122-131.(in Chinese)


Foundation:National Natural Science Foundation of China (31601014) ; Shenzhen Science and Technology Research Foundation(JCYJ20150525092940997; JYJC20140418091413587)
Corresponding author:Professor LIU Gang. E-mail: zjuliug@szu.edu.cn
Citation:WANG Juan, GONG Yanfen, LAI Yapeng, et al. The effect of RNA interference of MHR1 on cellulase activity in Myceliophthora thermophila[J]. Journal of Shenzhen University Science and Engineering, 2018, 35(1): 15-21.(in Chinese)
作者简介:王娟(1975—),女,深圳大学副教授、博士.研究方向:应用微生物学. E-mail:wangjuan@szu.edu.cn
引文:王娟,龚艳芬,来亚鹏,等.干扰MHR1对嗜热毁丝霉纤维素酶活性的影响[J]. 深圳大学学报理工版,2018,35(1):15-21.
更新日期/Last Update: 2017-12-22