香菇HMG-box转录因子lelcrp1基因的功能
作者:
基金项目:

公益性行业(农业)科研专项(201503137);湖北省技术创新重大项目(2016ABA100)


Function analysis of HMG-box transcription factor lelcrp1 in Lentinula edodes
Author:
  • 摘要
  • | |
  • 访问统计
  • |
  • 参考文献 [23]
  • |
  • 相似文献 [20]
  • | | |
  • 文章评论
    摘要:

    [目的]研究香菇(Lentinula edodes) HMG-box转录因子LELCRP1(Lentinula edodes lignocellulase genes regulation protein 1)在木质纤维素降解相关酶基因表达中的功能与作用。[方法]通过double-joint及同源重组方法构建lelcrp1基因RNAi载体,采用根癌农杆菌介导转化的方法转入香菇异核菌株W1菌丝中,筛选得到RNAi转化子,通过Southern杂交检测插入片段在菌株W1基因组中的拷贝数量。采用荧光定量PCR检测RNAi转化子木质纤维素降解酶基因表达水平变化,并在含有3.5 μg/mL潮霉素的MYG平板上测定RNAi转化子的菌丝生长速度。[结果]获得了4个lelcrp1基因表达水平与出发菌株W1相比显著下调6-7倍的RNAi转化子。Southern杂交结果显示,lelcrp1基因RNAi片段已成功整合至香菇菌株W1基因组内,并以单拷贝形式存在。对其中2个RNAi转化子的26个木质纤维素降解酶基因表达水平进行分析,发现其中9个纤维素酶基因、1个半纤维素酶基因、2个辅助酶AA9基因和1个锰过氧化物酶基因的表达水平均表现出明显的下调。平板生长试验表明,RNAi转化子菌丝生长速度均显著慢于出发菌株W1。[结论]通过RNAi技术成功抑制了香菇异核菌株中lelcrp1基因表达水平,并导致部分纤维素及木质素酶基因表达水平相应下调,首次发现HMG-box结构域的转录因子能调控木质纤维素降解相关酶基因表达。

    Abstract:

    [Objective] The objective of this study was to analyze the function of the HMG-box transcription factor LELCRP1 (Lentinula edodes lignocellulase genes regulation protein 1) in the expression of lignocellulase genes.[Methods] The RNAi vector of lelcrp1 was constructed by double-joint and homologous recombination, then transformed into the mycelia of Lentinula edodes heterotypic strain W1 by Agrobacterium tumefaciens-mediated transformation (ATMT). The copy number of the inserted fragment in the genome of strain W1 was detected by Southern blotting. Fluorescent quantitative PCR was used to detect the expression level of lignocellulase genes in RNAi transformants, and the mycelial growth rate was measured on MYG plates.[Results] We got four RNAi transformants which were significantly down-regulated by 6 to 7-fold compared with the original strain W1. The results of Southern blotting showed that the lelcrp1 gene fragment had been successfully integrated into the genome of Lentinula edodes in a single copy. Analysis of expression profiles of 26 lignocellulase genes in two RNAi transformants revealed that 9 cellulase genes, 1 hemicellulase gene, 2 auxiliary enzymes AA9 gene and 1 manganese peroxide gene were down-regulated significantly, compared to the original strain. The mycelial growth rate of RNAi transformants were significantly slower than the original strain.[Conclusion] The expression of lelcrp1 was silenced successfully by RNAi, resulting in down-regulation of the expression level of some cellulose and ligninase genes. The HMG-box domain transcription factor could regulate the expression of lignocellulase genes.

    参考文献
    [1] Royse DJ, Sanchez JE. Ground wheat straw as a substitute for portions of oak wood chips used in shiitake (Lentinula edodes) substrate formulae. Bioresource Technology, 2007, 98(11):2137-2141.
    [2] Znameroski EA, Coradetti ST, Roche CM, Tsai JC, Iavarone AT, Cate JHD, Glass NL. Induction of lignocellulose-degrading enzymes in Neurospora crassa by cellodextrins. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(16):6012-6017.
    [3] Nakazawa T, Izuno A, Kodera R, Miyazaki Y, Sakamoto M, Isagi Y, Honda Y. Identification of two mutations that cause defects in the ligninolytic system through an efficient forward genetics in the white-rot agaricomycete Pleurotus ostreatus. Environmental Microbiology, 2016, 19(1):261-272.
    [4] Štros M, Launholt D, Grasser KD. The HMG-box:a versatile protein domain occurring in a wide variety of DNA-binding proteins. Cellular and Molecular Life Sciences, 2007, 64(19/20):2590-2606.
    [5] Merz K, Hondele M, Goetze H, Gmelch K, Stoeckl U, Griesenbeck J. Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules. Genes & Development, 2008, 22(9):1190-1204.
    [6] Ray S, Grove A. The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends. Nucleic Acids Research, 2009, 37(19):6389-6399.
    [7] MacAlpine DM, Perlman PS, Butow RA. The high mobility group protein Abf2p influences the level of yeast mitochondrial DNA recombination intermediates in vivo. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(12):6739-6743.
    [8] Stillman DJ. Nhp6:a small but powerful effector of chromatin structure in Saccharomyces cerevisiae. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 2010, 1799(1/2):175-180.
    [9] Albert B, Colleran C, Léger-Silvestre I, Berger AB, Dez C, Normand C, Perez-Fernandez J, McStay B, Gadal O. Structure-function analysis of Hmo1 unveils an ancestral organization of HMG-Box factors involved in ribosomal DNA transcription from yeast to human. Nucleic Acids Research, 2013, 41(22):10135-10149.
    [10] Dequard-Chablat M, Alland C. Two copies of mthmg1, encoding a novel mitochondrial HMG-like protein, delay accumulation of mitochondrial DNA deletions in Podospora anserine. Eukaryot Cell, 2002, 1(4):503-513.
    [11] Ohm RA, de Jong JF, de Bekker C, Wösten HA, Lugones LG. Transcription factor genes of Schizophyllum commune involved in regulation of mushroom formation. Molecular Microbiology, 2011, 81(6):1433-1445.
    [12] Shi LL, van Peer AF, Guo L, Chen RL, Wang W, Yan JJ, Deng YJ, Xie BG. Agrobacterium-mediated transformation of an endogenous HMG-box transcription factor fvhom1 in Flammulina velutipes. Genomics and Applied Biology, 2014, 33(6):1268-1274. (in Chinese)施乐乐, van Peer AF, 郭丽, 陈仁良, 王威, 严俊杰, 邓优锦, 谢宝贵. 农杆菌介导一个内源HMG-box转录因子fvhom1转化金针菇. 基因组学与应用生物学, 2014, 33(6):1268-1274.
    [13] Cai YL, Gong YH, Liu W, Hu Y, Chen LF, Yan LL, Zhou Y, Bian YB. Comparative secretomic analysis of lignocellulose degradation by Lentinula edodes grown on microcrystalline cellulose, lignosulfonate and glucose. Journal of Proteomics, 2017, 163:92-101.
    [14] Cho JH, Lee SE, Chang WB, Cha JS. Agrobacteriummediated transformation of the winter mushroom, Flammulina velutipes. Mycobiology, 2006, 34(2):104-107.
    [15] Li C, Gong WB, Zhang L, Yang ZQ, Nong WY, Bian YB, Kwan HS, Cheung MK, Xiao Y. Association mapping reveals genetic loci associated with important agronomic traits in Lentinula edodes, shiitake mushroom. Frontiers in Microbiology, 2017, 8:237.
    [16] Chen LF, Gong YH, Cai YL, Liu W, Zhou Y, Xiao Y, Xu ZY, Liu Y, Lei XY, Wang GZ, Guo MP, Ma XL, Bian YB. Genome sequence of the edible cultivated mushroom Lentinula edodes (Shiitake) reveals insights into lignocellulose degradation. PLoS One, 2016, 11(8):e0160336.
    [17] Liu WZ, Xie YB, Ma JY, Luo XT, Nie P, Zuo ZX, Lahrmann U, Zhao Q, Zheng YY, Zhao Y, Xue Y, Ren J. IBS:an illustrator for the presentation and visualization of biological sequences. Bioinformatics, 2015, 31(20):3359-3361.
    [18] Yu JH, Hamari Z, Han KH, Seo JA, Reyes-Domínguez Y, Scazzocchio C. Double-joint PCR:a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genetics and Biology, 2004, 41(11):973-981.
    [19] Dörnte B, Kües U. Fast microwave-based DNA extraction from vegetative mycelium and fruiting body tissues of agaricomycetes for PCR amplification. Current Trends in Biotechnology & Pharmacy, 2013, 7(4):825-836.
    [20] Liu XM, Wu XL, Chen Q, Qiu ZH, Zhang JX, Huang CY. Effects of heat stress on Pleurotus eryngii mycelial growth and its resistance to Trichoderma asperellum. Mycosystema, 2017, 36(11):1566-1574. (in Chinese)刘秀明, 邬向丽, 陈强, 仇志恒, 张金霞, 黄晨阳. 高温胁迫对刺芹侧耳菌丝生长及其抗棘孢木霉能力的影响. 菌物学报, 2017, 36(11):1566-1574.
    [21] Fan XZ, Zhou Y, Xiao Y, Bian YB. Cloning and characterization of two allelic glyceraldehyde-3-phosphate dehydrogenase genes in Auricularia auricula-judae. World Journal of Microbiology and Biotechnology, 2014, 30(1):181-189.
    [22] Todd RB, Zhou M, Ohm RA, Leeggangers HA, Visser L, de Vries RP. Prevalence of transcription factors in ascomycete and basidiomycete fungi. BMC Genomics, 2014, 15:214.
    [23] Saloheimo A, Aro N, Ilmén M, Penttilä M. Isolation of the ace1 gene encoding a Cys2-His2 transcription factor involved in regulation of activity of the cellulase promoter cbh1 of Trichoderma reesei. The Journal of Biological Chemistry, 2000, 275(8):5817-5825.
    引证文献
    网友评论
    网友评论
    分享到微博
    发 布
引用本文

胡悦,蔡英丽,喻晶晶,周雁,李军平,边银丙,龚钰华. 香菇HMG-box转录因子lelcrp1基因的功能[J]. 微生物学报, 2018, 58(12): 2100-2109

复制
分享
文章指标
  • 点击次数:1195
  • 下载次数: 1534
  • HTML阅读次数: 2765
  • 引用次数: 0
历史
  • 收稿日期:2017-12-26
  • 最后修改日期:2018-01-29
  • 在线发布日期: 2018-12-05
文章二维码