基于转录-代谢联合分析哈茨木霉ACCC32527对NaCl胁迫的分子调节
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摘要
【目的】通过NaCl胁迫下哈茨木霉(Trichoderma harzianum)ACCC32527差异转录组和代谢组数据分析,挖掘关键功能型差异表达基因(DEG)及次级代谢产物。【方法】采用RNA-seq和GC-TOF-MS技术分别完成0(T1)、0.4(T2)、0.6(T3)mol·L~(-1) NaCl胁迫下哈茨木霉ACCC32527的差异表达基因和次级代谢产物检测,利用相关软件及数据库(GO、COG、KEGG pathway等)完成对差异表达基因(|log_2fold change)|>1&FDR<0.01)和次级代谢产物(P-value≤0.05&VIP>1)的筛选、注释和分类,并进行RT-qPCR验证。【结果】NaCl胁迫处理后,ACCC32527的生长受到抑制,繁殖速率明显降低,并在该条件下分别获得T1 vs T2和T1 vs T3比较组637、1 570个DEG,获得DEG的16种表达模式,包括9种上调表达模式(950 gene)、3种下调表达模式(662 gene)和4种不规则表达模式(309 gene),共有的GO功能分类为催化活性、结合、细胞器、细胞膜部分、细胞膜、细胞部分、细胞、单组织过程和代谢过程,且占主要比例,约为61%—94%,其中处理前后基因比例差异较大的分类为催化活性。KEGG代谢通路富集分析发现,不同NaCl浓度处理下的DEG富集到的代谢通路种类及数量存在较大差异,分别有225和535个差异基因富集于20条KEGG通路,包括代谢通路、抗生素的合成和次级代谢产物合成,其中T2和T3处理下核糖体的生物合成途径分别有12个和18个相关基因受到抑制。从NaCl胁迫下差异转录组中筛选出活性氧清除、离子转运和细胞壁及胞外结构等共73个相关基因,并且多数为上调表达基因。另外,差异代谢组学分析表明,T3处理下,胞内小分子代谢物出现明显变化,累积量增加的代谢产物有30种,包括氨基酸及其衍生物、糖类及其衍生物和醇类,而减少的代谢物有53种,涉及脂肪酸和有机酸等。其中,甘油是已知的真菌重要渗透保护物,T3处理下ACCC32527胞内甘油水平提高,可参与细胞的渗透调节,维持渗透压平衡。RT-qPCR验证了7个差异表达显著基因,虽然在表达量上存在一定的差异,但表达趋势与RNA-seq分析结果基本一致,表明转录组测序结果的可靠性。【结论】NaCl胁迫引起哈茨木霉ACCC32527大量基因及次级代谢产物变化,获得了与NaCl胁迫调节相关基因和代谢产物,这些机制共同作用减少盐离子对细胞的毒害作用,研究结果可为研究木霉菌的耐盐机理提供重要信息。
【Objective】The objective of this study is to mine the key functional differentially expressed genes(DEGs) and secondary metabolites by analyzing the data of Trichoderma harzianum ACCC32527 differential transcriptomes and metabolomes under NaCl stress.【Method】The transcriptomes and metabolomes of ACCC32527 with the treatment of 0(T1), 0.4(T2), 0.6(T3)mol·L~(-1) NaCl were performed by RNA-seq and GC-TOF-MS, respectively, and the annotation, screening and classification of DEGs(|log_2fold change|>1 & FDR<0.01) and secondary metabolites(P-value≤0.05 & VIP>1) were completed by related softwares and databases(GO, COG and KEGG pathway). Finally, RT-qPCR was conducted to validate RNA-seq data.【Result】In this study, after Na Cl stress treatments, the growth of ACCC32527 was inhibited and the reproductive rate was significantly decreased, 637 and1 570 DEGs were screened from the T1 vs T2 and T1 vs T3 comparison groups, respectively. Trend analysis result showed that 1 921 DEGs were assigned to 16 significant expression patterns, including 9 up-regulated trends(950 genes), 3 down-regulated trends(662 genes) and 4 irregular trends(309 genes). GO enrichment analysis result showed that each tread was shared by catalytic activity,binding, organelle, cell membrane part, cell membrane, cell part, cell, single-organism process and metabolic process, and accounted for 61%-94%. Among them, the classification with great difference in gene proportion before and after treatment was catalytic activity. The enrichment analysis of KEGG metabolic pathway displayed that the classification and numbers of pathways treated with different NaCl concentrations were significantly different, there were 225 and 535 DEGs enriched in 20 KEGG pathways,respectively, including metabolic pathways, biosynthesis of antibiotics, biosynthesis of secondary metabolites and so on. Among them, 12 and 18 related genes of ribosome biosynthesis pathway were suppressed under T2 and T3 treatments, respectively. A total of73 genes were screened from the DEGs including ROS scavenging, ion transport and cell wall structure, and most of them were up-regulated. In addition, intracellular small molecule metabolites showed significant change in response to NaCl stress, and there were 30 metabolites with increased accumulation, including amino acids and their derivatives, sugars and their derivatives and alcohols, while 53 metabolites were reduced including fatty acids and organic acids. Among them, glycerol, a known important osmoregulation substance of fungi, was increased under T3 treatment, which may be involved in the process of cell osmotic adjustment. RT-qPCR verified the expression changes of 7 DEGs, which were consistent with the RNA-seq analysis, indicating the reliability of transcriptome sequencing results.【Conclusion】Under NaCl stress, a large number of genes and secondary metabolites of T. harzianum ACCC32527 were changed, which were related to NaCl stress regulation. These processes act together to reduce the toxic effect to the cells, and the results can provide important information for the study of salt-tolerant mechanism of Trichoderma spp..
【Objective】The objective of this study is to mine the key functional differentially expressed genes(DEGs) and secondary metabolites by analyzing the data of Trichoderma harzianum ACCC32527 differential transcriptomes and metabolomes under NaCl stress.【Method】The transcriptomes and metabolomes of ACCC32527 with the treatment of 0(T1), 0.4(T2), 0.6(T3)mol·L~(-1) NaCl were performed by RNA-seq and GC-TOF-MS, respectively, and the annotation, screening and classification of DEGs(|log_2fold change|>1 & FDR<0.01) and secondary metabolites(P-value≤0.05 & VIP>1) were completed by related softwares and databases(GO, COG and KEGG pathway). Finally, RT-qPCR was conducted to validate RNA-seq data.【Result】In this study, after Na Cl stress treatments, the growth of ACCC32527 was inhibited and the reproductive rate was significantly decreased, 637 and1 570 DEGs were screened from the T1 vs T2 and T1 vs T3 comparison groups, respectively. Trend analysis result showed that 1 921 DEGs were assigned to 16 significant expression patterns, including 9 up-regulated trends(950 genes), 3 down-regulated trends(662 genes) and 4 irregular trends(309 genes). GO enrichment analysis result showed that each tread was shared by catalytic activity,binding, organelle, cell membrane part, cell membrane, cell part, cell, single-organism process and metabolic process, and accounted for 61%-94%. Among them, the classification with great difference in gene proportion before and after treatment was catalytic activity. The enrichment analysis of KEGG metabolic pathway displayed that the classification and numbers of pathways treated with different NaCl concentrations were significantly different, there were 225 and 535 DEGs enriched in 20 KEGG pathways,respectively, including metabolic pathways, biosynthesis of antibiotics, biosynthesis of secondary metabolites and so on. Among them, 12 and 18 related genes of ribosome biosynthesis pathway were suppressed under T2 and T3 treatments, respectively. A total of73 genes were screened from the DEGs including ROS scavenging, ion transport and cell wall structure, and most of them were up-regulated. In addition, intracellular small molecule metabolites showed significant change in response to NaCl stress, and there were 30 metabolites with increased accumulation, including amino acids and their derivatives, sugars and their derivatives and alcohols, while 53 metabolites were reduced including fatty acids and organic acids. Among them, glycerol, a known important osmoregulation substance of fungi, was increased under T3 treatment, which may be involved in the process of cell osmotic adjustment. RT-qPCR verified the expression changes of 7 DEGs, which were consistent with the RNA-seq analysis, indicating the reliability of transcriptome sequencing results.【Conclusion】Under NaCl stress, a large number of genes and secondary metabolites of T. harzianum ACCC32527 were changed, which were related to NaCl stress regulation. These processes act together to reduce the toxic effect to the cells, and the results can provide important information for the study of salt-tolerant mechanism of Trichoderma spp..
引文
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[22]WILLI J,KUPFER P,EVEQUOZ D,FERNANDEZ G,KATZ A,LEUMANN C,POLACEK N.Oxidative stress damages rRNA inside the ribosome and differentially affects the catalytic center.Nucleic Acids Research,2018,46(4):1945-1957.
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[24]HASE Y,TARUSAWA T,MUTO A,HIMENO H.Impairment of ribosome maturation or function confers salt resistance on Escherichia coli cells.PLoS ONE,2013,8(5):e65747.
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[2]ASLAM R,BOSTAN N,NABGHA A,MARIA M,SAFDAR W.Acritical review on halophytes:Salt tolerant plants.Journal of Medicinal Plants Research,2011,5(33):7108-7118.
[3]UPADHYAY S K,SINGH D P.Effect of salt-tolerant plant growth-promoting rhizobacteria on wheat plants and soil health in a saline environment.Plant Biology,2015,17(1):288-293.
[4]PATEL R R,PATEL D D,THAKOR P,PATEL B,THAKKAR V R.Alleviation of salt stress in germination of Vigna radiata L.by two halotolerant Bacilli sp.isolated from saline habitats of Gujarat.Plant Growth Regulation,2015,76(1):51-60.
[5]KHOMARI S,GOLSHAN-DOUST S,SEYED-SHARIFI R,DAVARIM.Improvement of soybean seedling growth under salinity stress by biopriming of high-vigour seeds with salt-tolerant isolate of Trichoderma harzianum.New Zealand Journal of Crop and Horticultural Science,2018,46(2):117-132.
[6]SCHUSTER A,SCHMOLL M.Biology and biotechnology of Trichoderma.Applied Microbiology and Biotechnology,2010,87(3):787-799.
[7]RAWAT L,SINGH Y,SHUKLA N,KUMAR J.Salinity tolerant Trichoderma harzianum reinforces Na Cl tolerance and reduces population dynamics of Fusarium oxysporum f.sp.ciceri in chickpea(Cicer arietinum L.)under salt stress conditions.Archives of Phytopathology and Plant Protection,2013,46(12):1442-1467.
[8]HASHEM A,ABD-ALLAH E F,ALQARAWI A A,AL HUQAIL A A,EGAMBERDIEVA D.Alleviation of abiotic salt stress in Ochradenus baccatus(Del.)by Trichoderma hamatum(Bonord.)Bainier.Journal of Plant Interactions,2014,9(1):857-868.
[9]邓勋,宋小双,尹大川,宋瑞清.盐胁迫对2株深色有隔内生真菌(DSE)生长及营养代谢的影响.中南林业科技大学学报,2015,35(5):1-8.DENG X,SONG X S,YIN D C,SONG R Q.Effects of salt stress on growth and nutritional metabolism of two dark septate endophyte(DSE).Journal of Central South University of Forestry&Technology,2015,35(5):1-8.(in Chinese)
[10]SAMAPUNDO S,AMPOFO-ASIAMA J,ANTHIERENS T,XHAFERI R,VAN BREE I,SZCZEPANIAK S,GOEMAERE O,STEEN L,DHOOGE M,PAELINCK H,DEWETTINCK K,DEVLIEGHERE F.Influence of Na Cl reduction and replacement on the growth of Lactobacillus sakei in broth,cooked ham and white sauce.International Journal of Food Microbiology,2010,143(1/2):9-16.
[11]ZEHRA A,DUBEY M K,MEENA M,UPADHYAY R S.Effect of different environmental conditions on growth and sporulation of some Trichoderma species.Journal of Environmental Biology,2017,38:197-203.
[12]RUPPEL S,FRANKEN P,WITZEL K.Properties of the halophyte microbiome and their implications for plant salt tolerance.Functional Plant Biology,2013,40(8/9):940-951.
[13]DURAN R,CARY J W,CALVO A M.Role of the osmotic stress regulatory pathway in morphogenesis and secondary metabolism in filamentous fungi.Toxins,2010,2(4):367-381.
[14]DELGADO-JARANA J,SOUSA S,GONZALEZ F,REY M,LIOBELL A.ThHog1 controls the hyperosmotic stress response in Trichoderma harzianum.Microbiology,2006,152(6):1687-1700.
[15]YANG L,YANG Q,SUN K,TIAN Y,LI H.Agrobacterium tumefaciens-mediated transformation of SOD gene to Trichoderma harzianum.World Journal of Microbiology and Biotechnology,2010,26(2):353-358.
[16]GAO J X,WANG Q Q,SUN J N,HE A,CHEN J.Biological role of the superoxide dismutase Ta SOD on vegetative growth,stress response,and antagonism in Trichoderma asperellum.Australasian Plant Pathology,2018,47(6):623-627.
[17]崔西苓,李世贵,杨佳,樊祥臣,顾金刚.耐盐碱抗烟草黑胫病木霉菌株的筛选与鉴定.中国农业科技导报,2014,16(3):81-89.CUI X L,LI S G,YANG J,FAN X C,GU J G.Screening and identification of saline-alkali tolerant and tobacco black shank resistant Trichoderma.Journal of Agricultural Science and Technology,2014,16(3):81-89.(in Chinese)
[18]杨俊慧,郑岚,马耀宏,张利群,杨艳.马铃薯中还原糖不同测定方法的比较.食品研究与开发,2011,32(6):104-108.YANG J H,ZHENG L,MA Y H,ZHANG L Q,YANG Y.Comparison of different determination of reducing sugar in potato.Food Research and Development,2011,32(6):104-108.(in Chinese)
[19]王丽荣,蒋细良,ESTIFANOS T,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.中国生物防治学报,2017,33(1):103-113.WANG L R,JIANG X L,ESTIFANOS T,DING J,MA J,CHEN X L,LI X Y,LI M.Transcriptome analysis of Trichoderma harzianum Th-33 under copper stress.Chinese Journal of Biological Control,2017,33(1):103-113.(in Chinese)
[20]WU Q,NI M,WANG G S,LIU Q Q,YU M X,TANG J.Omics for understanding the tolerant mechanism of Trichoderma asperellum TJ01 to organophosphorus pesticide dichlorvos.BMC Genomics,2018,19:596.
[21]CHOUDHURY F K,RIVERO R M,BLUMWALD E,MITTLER R.Reactive oxygen species,abiotic stress and stress combination.The Plant Journal,2017,90(5):856-867.
[22]WILLI J,KUPFER P,EVEQUOZ D,FERNANDEZ G,KATZ A,LEUMANN C,POLACEK N.Oxidative stress damages rRNA inside the ribosome and differentially affects the catalytic center.Nucleic Acids Research,2018,46(4):1945-1957.
[23]LI J,GIESY J P,YU L,LI G Y,LIU C S.Effects of tris(1,3-dichloro-2-propyl)phosphate(TDCPP)in Tetrahymena thermophila:targeting the ribosome.Scientific Reports,2015,5:10562.
[24]HASE Y,TARUSAWA T,MUTO A,HIMENO H.Impairment of ribosome maturation or function confers salt resistance on Escherichia coli cells.PLoS ONE,2013,8(5):e65747.
[25]周志刚,包虹,欧阳珑玲.缺刻缘绿藻溶血磷脂酰乙醇胺酰基转移酶(LPEAT)的基因克隆与特征分析.中国水产科学,2018,25(2):251-262.ZHOU Z G,BAO H,OUYANG L L.Cloning and characterization of a gene encoding lysophosphatidylethanolamine acyltransferase(LPEAT)in the green microalga Myrmecia incise Reisigl.Journal of Fishery Sciences of China,2018,25(2):251-262.(in Chinese)
[26]BERRY S,ESPER B,KARANDASHOVA I,TEUBER M,ELANSKASA I,ROGNER M,HAGEMANN M.Potassium uptake in the unicellular cyanobacterium Synechocystis sp.strain PCC 6803mainly depends on a Ktr-like system encoded by slr1509(ntpJ).FEBSLetters,2003,548(1/3):53-58.
[27]TEJERA N A,CAMPOS R,SANJUAN J,LIUCH C.Nitrogenase and antioxidant enzyme activities in Phaseolus vulgaris nodules formed by Rhizobium tropici isogenic strains with varying tolerance to salt stress.Journal of Plant Physiology,2004,161(3):329-338.
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