铜胁迫下的哈茨木霉Th-33转录组分析

doi:10.16409/j.cnki.2095-039x.2017.01.015

摘要/Abstract

摘要： 采用Hiseq2500高通量测序平台完成了铜胁迫和对照条件下哈茨木霉Th-33的转录组测序，共检测出差异表达基因891条，其中上调表达基因570条，下调表达基因321条。GO富集分析显示，共有438个差异表达基因参与到673个不同的功能亚类中；KEGG代谢途径分析显示，共有254个差异表达基因归到167个代谢通路中，其中涉及基因最多的是对氨基苯甲酸甲酯降解和氯烷烃、烯烷烃降解通路。通过对差异表达基因和代谢通路分析，推导出菌株Th-33细胞内可能的铜胁迫应答途径。铜胁迫下，Cu²⁺还原为Cu⁺、铜转运蛋白（copper transporter，Ctr）的转运途径受到抑制，铜离子可能一部分通过吞饮的作用进入哈茨木霉Th-33细胞，可能通过与谷胱甘肽（GST）结合，或通过P-type ATP酶的外排作用将铜离子向质膜外运输以减少对细胞的毒害作用。

关键词: 铜胁迫, 转录组测序, 哈茨木霉, 应答

Abstract: The transcripts of Trichoderma harzianum Th-33 under copper stress and normal conditions were sequenced on a Hiseq2500 instrument. A total of 891 differentially expressed genes (DEGs) were found between the two transcripts, of which 570 genes were up-regulated and 321 genes were down-regulated in expression. GO analysis suggested that total of 438 differentially expressed genes were categorized into 673 functional groups. KEGG analysis revealed that total of 254 genes were annotated in 167 individual pathways and these unigenes were significantly enriched in various known aminobenzoate degradation and chloroalkane degradation. According to the differentially expressed genes and metabolic pathways of T. harzianum Th-33 under copper stress, the copper transport and metabolism pattern in T. harzianum Th-33 were different from that of yeast and T. reesei. Pathways of reducing of copper ions and Ctr transporting were inhibited by copper stress, and part of copper may enter T. harzianum Th-33 cells by endocytosis. It was speculated that copper transferred in T. harzianum Th-33 by combining with glutathione, or transported to the plasma membrane by P-type ATPase.

Key words: copper stress, transcriptome sequencing, Trichoderma harzianum, response

中图分类号:

S476

王丽荣, 蒋细良, Estifanos Tsegaye, 丁洁, 马景, 陈孝利, 李昕玥, 李梅. 铜胁迫下的哈茨木霉Th-33转录组分析[J]. 中国生物防治学报, 2017, 33(1): 103-113.

WANG Lirong, JIANG Xiliang, ESTIFANOS Tsegaye, DING Jie, MA Jing, CHEN Xiaoli, LI Xinyue, LI Mei. Transcriptome Analysis of Trichoderma harzianum Th-33 under Copper Stress[J]. journal1, 2017, 33(1): 103-113.

参考文献

[1] Vance E D, Brookes P C, Jenkinson D S. An extraction method for measuring soil microbial biomass C[J]. Soil Biology and Biochemistry, 1987, 19(6):703-707.
[2] Bhattacharyya S, Pal T K, Basumajumdar A. Modulation of enzyme activities of a lead-adapted strain of Rhizopus arrhizus during bioaccumulation of lead[J]. Folia Microbiologica, 2009, 54(6):505-508.
[3] Weindling R. Trichoderma lignorum as a parasite of other soil fungi[J]. Phytopathology, 1932, 22(8):837-845.
[4] de Santiago A, Quintero J M, Avilés M, et al. Effect of Trichoderma asperellum strain T34 on iron, copper, manganese, and zinc uptake by wheat grown on a calcareous medium[J]. Plant and Soil, 2011, 342(1/2):97-104.
[5] Castellanos F, Schmoll M, Martínez P, et al. Crucial factors of the light perception machinery and their impact on growth and cellulase gene transcription in Trichoderma reesei[J]. Fungal Genetics and Biology, 2010, 47(5):468-476.
[6] Casas-Flores S, Rios-Momberg M, Bibbins M, et al. BLR-1 and BLR-2, key regulatory elements of photoconidiation and mycelial growth in Trichoderma atroviride[J]. Microbiology, 2004, 150(11):3561-3569.
[7] 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.
[8] Adams T H, Wieser J K, Yu J. Asexual sporulation in Aspergillus nidulans[J]. Microbiology and Molecular Biology Reviews, 1998, 62(1):35-54.
[9] 张黛静, 王多多, 董文, 等. 铜胁迫下小麦幼根转录组学及蛋白质组学研究[J]. 河南农业科学, 2015, 44(4):31-35.
[10] Suresh K, Subramanyam C. Polyphenols are involved in copper binding to cell walls of Neurospora crassa[J]. Journal of Inorganic Biochemistry, 1998, 69(4):209-215.
[11] Mullen M D, Wolf D C, Beveridge T J, et al. Sorption of heavy metals by the soil fungi Aspergillus niger and Mucor rouxii[J]. Soil Biology and Biochemistry, 1992, 24(2):129-135.
[12] Klis F M, Mol P, Hellingwerf K, et al. Dynamics of cell wall structure in Saccharomyces cerevisiae[J]. FEMS Microbiology Reviews, 2002, 26(3):239-256.
[13] Strange J, Macnair M R. Evidence for a role for the cell membrane in copper tolerance of Mimulus guttatus Fischer ex DC.[J]. New Phytologist, 1991, 119(3):383-388.
[14] 冀颖, 李梅, 田云龙, 等. 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.
[15] Zhao S, Liu Q, Qi Y, et al. Responses of root growth and protective enzymes to copper stress in turfgrass[J]. Acta Biologica Cracoviensia Series Botanica, 2010, 52(2):7-11.
[16] Ajayan K V, Selvaraju M. Heavy metal induced antioxidant defense system of green microalgae and its effective role in phycoremediation of tannery effluent[J]. Pakistan Journal of Biological Sciences, 2012, 15(22):1056.
[17] 马占强, 赵龙飞, 王莉, 等. 铜胁迫对苜蓿中华根瘤菌抗氧化酶系的影响[J]. 农业环境科学学报, 2011, 30(6):1058-1063.
[18] Abdel-Ghany S E, Pilon M. MicroRNA-mediated systemic down-regulation of copper protein expression in response to low copper availability in Arabidopsis[J]. Journal of Biological Chemistry, 2008, 283(23):15932-15945.
[19] Gao J, Tao L, Yu X. Gene expression and activities of SOD in cucumber seedlings were related with concentrations of Mn²⁺, Cu²⁺, or Zn²⁺ under low temperature stress[J]. Agricultural Sciences in China, 2009, 8(6):678-684.
[20] 袁敏, 铁柏清, 唐美珍. 重金属单一污染对龙须草叶绿素含量和抗氧化酶系统的影响[J]. 土壤通报, 2005, 36(6):929-932.
[21] 熊思, 林爱军, 宋亮, 等. 铜污染对玉米幼苗的毒性及其机制研究[J]. 北京化工大学学报(自然科学版), 2013, 40(6):82-87.
[22] Zhu Y L, Pilon-Smits E A, Jouanin L, et al. Overexpression of glutathione synthetase in Indian mustard enhances cadmium accumulation and tolerance[J]. Plant Physiology, 1999, 119(1):73-80.
[23] Georgatsou E, Mavrogiannis L A, Fragiadakis G S, et al. The yeast Fre1p/Fre2p cupric reductases facilitate copper uptake and are regulated by the copper-modulated Mac1p activator[J]. Journal of Biological Chemistry, 1997, 272(21):13786-13792.
[24] Dancis A, Yuan D S, Haile D, et al. Molecular characterization of a copper transport protein in S. cerevisiae:an unexpected role for copper in iron transport[J]. Cell, 1994, 76(2):393-402.
[25] Rees E M, Thiele D J. Identification of a vacuole-associated metalloreductase and its role in Ctr2-mediated intracellular copper mobilization[J]. Journal of Biological Chemistry, 2007, 282(30):21629-21638.
[26] Hassett R, Kosman D J. The Fe (Ⅱ) permease Fet4p functions as a low affinity copper transporter and supports normal copper trafficking in Saccharomyces cerevisiae[J]. Biochemical Journal, 2000, 351(2):477-484.
[27] Horng Y, Cobine P A, Maxfield A B, et al. Specific copper transfer from the Cox17 metallochaperone to both Sco1 and Cox11 in the assembly of yeast cytochrome C oxidase[J]. Journal of Biological Chemistry, 2004, 279(34):35334-35340.
[28] Arnesano F, Banci L, Bertini I, et al. Characterization of the binding interface between the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase[J]. Journal of Biological Chemistry, 2001, 276(44):41365-41376.
[29] 傅科鹤. 里氏木霉铜代谢相关基因克隆与功能分析[D]. 上海:上海交通大学, 2013.
[30] 付世新, 王瑶, 罗春海, 等. Ctr1基因mRNA转录水平与细胞内铜离子浓度的相关性[J]. 中国生物制品学杂志, 2010, 23(5):482-484.
[31] Huffman D L, O'Halloran T V. Function, structure, and mechanism of intracellular copper trafficking proteins[J]. Annual Review of Biochemistry, 2001, 70(1):677-701.
[32] Greco M A, Hrab D I, Magner W, et al. Cu, Zn superoxide dismutase and copper deprivation and toxicity in Saccharomyces cerevisiae[J]. Journal of Bacteriology, 1990, 172(1):317-325.
[33] Pierson H, Lutsenko S, Tümer Z. Copper Metabolism, ATP7A and Menkes Disease[Z]. John Wiley and Sons Ltd, 2015.