基于转录组分析的哈茨木霉thga3基因功能研究

doi:10.16409/j.cnki.2095-039x.2018.01.015

摘要/Abstract

摘要： 哈茨木霉Trichoderma harzianum Th-33的Ⅲ型Gα亚基Thga3可能参与调控木霉菌的生长、产孢、几丁质酶活性、疏水性等生物学过程。为进一步明确其功能，本研究采用Hiseq2500（Illumina）测序平台，对野生菌Th-33和thga3基因敲除突变株△thga3进行转录组测序，分析其G蛋白信号系统、细胞壁降解酶类和产孢相关基因的差异表达。与野生菌相比，△thga3有3个G蛋白偶联受体基因发生下调表达，3个几丁质酶基因以及产孢调控基因brlA下调表达，1个G蛋白信号调控蛋白RGS1上调表达。推测thga3可能通过调控brlA的表达正调控产孢；通过调控疏水蛋白基因Tha_09745的表达调控菌丝疏水性。此外，通过调控几丁质酶基因的表达、G蛋白信号网络中其他蛋白的表达，影响菌株的拮抗和重寄生能力以及其他生物学特性。本研究为进一步探索G蛋白信号途径调控木霉菌生防特性及其机制奠定了基础。

关键词: 哈茨木霉, 转录组测序, 差异表达基因, Thga3

Abstract: thga3 gene encoding a type Ⅲ G protein α subunit Thga3 in Trichoderma harzianum Th-33 may participated in the regulation of growth, spore production, mycoparasitism, hydrophobic and chitinase activity. In order to clarify its function, Illumina RNA-seq technology (RNA-seq) was used to analyze the differentially expressed genes (DEGs) between the wild-type strain Th-33 and thga3 knockout mutant △thga3, and the expression of genes related to the G protein signaling pathways, cell wall degradation enzymes, and the spore production were analysed. Comparing to wild Th-33, three DEGs of G protein receptor proteins, three chitinase genes, one sporulation regulatory gene brlA encoding a nucleic acid binding protein and one hydrophobin gene Tha_09745 were down-regulated, and one G protein signal transduction protein gene coding RGS1 was up-regulated in △thga3. It was hypothesized that thga3 may regulate sporulation by regulating the expression of brlA, regulate the mycelial hydrophobicity by regulating the expression of Tha_09745, and regulate the chitinase activity and affect the expression of other proteins in the G protein signaling network, which may affect the antagonism and mycoparasitism, as well as other biological characteristics. This study lays a foundation for further exploration on the regulation of G protein signaling pathway in Trichoderma biocontrol characteristics and its mechanism.

Key words: Trichoderma harzianum, transcriptome sequencing, differentially expressed genes, Thga3

中图分类号:

S476

丁洁, 蒋细良, 梅杰, 孙青, 李梅. 基于转录组分析的哈茨木霉thga3基因功能研究[J]. 中国生物防治学报, 2018, 34(1): 124-132.

DING Jie, JIANG Xiliang, MEI Jie, SUN Qing, LI Mei. Functions of Thga3 Gene in Trichoderma harzianum Based on Transcriptome Analysis[J]. journal1, 2018, 34(1): 124-132.

参考文献

[1] 孙青. 基于转录组分析的哈茨木霉Thga1基因功能研究[D]. 北京:中国农业科学院, 2015.
[2] Baroncelli R, Piaggeschi G, Fiorini L, et al. Draft whole-genome sequence of the biocontrol agent Trichoderma harzianum T6776[J]. Genome Announcements. 2015, 3(3):e00647-15.
[3] 李利, 陈莎, 毛涛. 丝状真菌G蛋白信号途径的研究进展[J]. 微生物学通报, 2013, 40(8):1493-1507.
[4] Zeilinger S, Reithner B, Scala V, et al. Signal transduction by Tga3, a novel G protein alpha subunit of Trichoderma atroviride[J]. Applied and Environmental Microbiology, 2005, 71(3):1591-1597.
[5] Yu H Y, Seo J A, Kim J E, et al. Functional analyses of heterotrimeric G protein G alpha and G beta subunits in Gibberella zeae[J]. Microbiology, 2008, 154(2):392-401.
[6] Hu Y, Liu G, Li Z, et al. G protein-cAMP signaling pathway mediated by PGA3 plays different roles in regulating the expressions of amylases and cellulases in Penicillium decumbens[J]. Fungal Genetics and Biology, 2013, 58-59(5):62-70.
[7] Mukesh S, Vipul K, Mohammad S, et al. Trichoderma-a potential and effective bio fungicide and alternative source against notable phytopathogens:A review[J]. African Journal of Agricultural Research, 2016, 11(5):310-316.
[8] Vinale F, Ghisalberti E L, Sivasithamparam K, et al. Factors affecting the production of Trichoderma harzianum secondary metabolites during the interaction with different plant pathogens[J]. Letters Applied Microbiology, 2009, 48(6):705-711.
[9] Mukherjee P K, Latha J, Hadar R, et al. Role of two G-protein alpha subunits, TgaA and TgaB, in the antagonism of plant pathogens by Trichoderma virens[J]. Applied and Environmental Microbiology, 2004, 70(1):542-549.
[10] Komonzelazowska M, Neuhof T, Dieckmann R, et al. Formation of atroviridin by Hypocrea atroviridis is conidiation associated and positively regulated by blue light and the G protein GNA3[J]. Eukaryotic Cell, 2008, 6(12):2332-2342.
[11] Schmoll M, Esquivelnaranjo E U, Herreraestrella A, et al. Trichoderma in the light of day-physiology and development[J]. Fungal Genetics and Biology, 2010, 47(11):909-916.
[12] Martinez D, Berka R M, Henrissat B, et al. Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina)[J]. Nature biotechnology, 2008, 26(5):553-560.
[13] Bradford J R, Hey Y, Yates T, et al. A comparison of massively parallel nucleotide sequencing with oligonucleotide microarrays for global transcription profiling[J]. BMC Genomics, 2010, 11(1):282.
[14] Silva R D N, Steindorff A S, Ulhoa C J, et al. Involvement of G-alpha protein GNA3 in production of cell wall-degrading enzymes by Trichoderma reesei (Hypocrea jecorina) during mycoparasitism against Pythium ultimum[J]. Biotechnology Letters, 2009, 31(4):531-536.
[15] Muller P Y, Janovjak H, Miserez A R, et al. Processing of gene expression data generated by quantitative real-time RT-PCR[J]. Biotechniques, 2002, 32(6):1372-1374, 1376, 1378-1379.
[16] Esquivel Naranjo E U, García Esquivel M, Medina Castellanos E, et al. A Trichoderma atroviride stress-activated MAPK pathway integrates stress and light signals[J]. Molecular Microbiology, 2016, 100(5):860-876.
[17] Garzia A, Etxebeste O, Herrero-García E, et al. The concerted action of bZip and cMyb transcription factors FlbB and FlbD induces brlA expression and asexual development in Aspergillus nidulans[J]. Molecular Microbiology, 2010, 75(5):1314-1324.
[18] Adams T H, Boylan M T, Timberlake W E, et al. brlA is necessary and sufficient to direct conidiophore development in Aspergillus nidulans[J]. Cell, 1988, 54(3):353-362.
[19] Lafon A, Han K, Seo J, et al. G-protein and cAMP-mediated signaling in aspergilli:a genomic perspective[J]. Fungal Genetics and Biology, 2006, 43(7):490-502.
[20] 刘增亮, 李梅, 张玉杰. 哈茨木霉Thga1基因敲除突变株对几种植物病原真菌的离体拮抗作用[J]. 中国生物防治学报, 2014, 30(3):370-375.
[21] 刘增亮. 哈茨木霉Th-33 Thga1基因的克隆及功能研究[D]. 北京:中国农业科学院, 2013.
[22] Yu J H. Heterotrimeric G protein signaling and RGSs in Aspergillus nidulans[J]. Journal of Microbiology, 2006, 44(2):145-154.
[23] 彭陈, 陈洪亮, 张玉琼. 稻瘟菌附着胞形成和发育的研究进展[J]. 微生物学通报, 2011(8):1270-1277.
[24] Breakspear A, Momany M. Aspergillus nidulans conidiation genes dewA, fluG, and stuA are differentially regulated in early vegetative growth[J]. Eukaryotic Cell, 2007, 6(9):1697-1700.
[25] 鲍龙飞. 草酸青霉孢子发生和纤维素酶基因表达共调控机制研究[D]. 山东:山东大学, 2014.
[26] Wösten H A B, Vocht M L D. Hydrophobins, the fungal coat unravelled[J]. Biochimica et Biophysica Acta (BBA)-Reviews on Biomembranes, 2000, 1469(2):79-86.
[27] Linder M B, Szilvay G R, Nakarisetälä T, et al. Hydrophobins:the protein-amphiphiles of filamentous fungi[J]. FEMS Microbiology Reviews, 2005, 29(5):877-896.
[28] Bayry J, Aimanianda V, Guijarro J I, et al. Hydrophobins-unique fungal proteins[J]. PLoS Pathogens, 2012, 8(5):e1002700.
[29] 黄刚. 轮枝镰孢(Fusarium verticillioide)五种疏水蛋白的功能研究[D]. 山东:山东农业大学, 2013.
[30] Dubey M K, Dan F J, Karlsson M. Hydrophobins are required for conidial hydrophobicity and plant root colonization in the fungal biocontrol agent Clonostachys rosea[J]. BMC Microbiology, 2014, 14(1):1-14.
[31] Sun Q, Jiang X, Pang L, et al. Functions of thga1 gene in Trichoderma harzianum based on transcriptome analysis[J]. Biomed Research International, 2016(2016):1-9.
[32] 牛静, 李梅, 范亮波. 哈茨木霉caf类似基因ThCon1的克隆及功能初探[J]. 中国生物防治学报, 2013, 29(1):89-96.
[33] 冀颖, 李梅, 田云龙. 哈茨木霉几丁质合酶基因ThChsC的克隆及序列分析[J]. 中国农业科学, 2011, 44(17):3537-3546.