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林业科学 ›› 2019, Vol. 55 ›› Issue (8): 95-105.doi: 10.11707/j.1001-7488.20190811

• 论文与研究报告 • 上一篇    下一篇

偏肿革裥菌在木质环境下转录组构建与相关基因表达分析

赵清泉1, 池玉杰1, 张健1, 冯连荣1,2   

  1. 1. 东北林业大学林学院 哈尔滨 150040;
    2. 辽宁省杨树研究所 盖州 115213
  • 收稿日期:2018-08-10 修回日期:2019-01-17 发布日期:2019-09-05
  • 基金资助:
    中央高校基本科研业务费专项资金资助(2572017AA17);中央高校基本科研业务费科技平台持续发展专项资金资助(2572018CP05);黑龙江省自然科学基金项目(2016006)。

Transcriptome Construction and Related Gene Expression Analysis of Lenzites gibbosa in Woody Environment

Zhao Qingquan1, Chi Yujie1, Zhang Jian1, Feng Lianrong1,2   

  1. 1. School of Forestry, Northeast Forestry University Harbin 150040;
    2. Liaoning Provincial Institute of Poplar Gaizhou 115213
  • Received:2018-08-10 Revised:2019-01-17 Published:2019-09-05

摘要: [目的]对白腐菌偏肿革裥菌在木质和非木质环境下的转录组进行测序,为白腐菌降解木材的机制研究提供支持。[方法]采用高通量测序技术对木屑和非木屑处理条件下的菌丝样本进行转录组测序。利用eggNOG、GO、KEGG等数据库对转录本进行注释和比较分析,预测和筛选出偏肿革裥菌与木材降解有关的基因。应用荧光定量PCR (qPCR)检测mnp2等11个与木质素降解相关的差异基因在木屑处理5天条件下的表达量,结合转录组数据对这些基因的表达量变化趋势进行分析。[结果]偏肿革裥菌转录组测序共得到38.9 Gb Clean Data,各样品Clean Data平均达到6.49 Gb,各样品的Reads与参考基因组的比对效率在71.23%~74.25%之间。使用edgeR软件进行差异表达分析,得到差异表达基因898个,其中上调351个、下调547个。差异基因被注释到GO数据库的有251个,注释到KEGG数据库的有223个,注释到eggNOG数据库的有704个。eggNOG分析表明,差异基因表达多聚集在能量产生和转换、转录后修饰、蛋白质代谢、伴侣关系、次生代谢物的生物合成、碳水化合物的运输和分解代谢等功能分类下。GO分析表明,显著性富集与频率较高的生物过程是丙酮酸代谢过程、类异戊二烯生物合成过程、天冬氨酸家族氨基酸代谢过程和木质素分解过程。在KEGG通路分析中,差异基因分布到86个不同的生物途径,差异基因显著富集的代谢通路主要为:碳代谢、柠檬酸循环、芳香化合物降解、乙醛酸代谢。qPCR结果表明在木屑处理条件下基因mnp2mnp3lip9lip2laccase1nadp的表达量明显上调; mnp10 smrpcyp450-1GroES1GroES2的表达量明显下调,qPCR与转录组测序结果在表达量差异倍数上存在较小偏差,但整体趋势一致,可以证明转录组测序结果是正确可靠的。[结论]偏肿革裥菌降解木材与碳代谢、芳香化合物降解等通路密切相关;与木质素分解等生物过程密切相关。根据基因功能注释的结果得到11个与白腐菌降解木质素相关的重要差异表达基因。

关键词: 偏肿革裥菌, 白腐菌, 转录组, 木质素, 降解

Abstract: [Objective] The aim of this study is to provide a support for the study on the mechanism of wood degradation by Lenzites gibbosa (white-rot fungi) by sequencing the transcriptomes of white-rot fungus under woody and non-woody environments.[Method] High-throughput sequencing technology was used to sequence transcriptomes of mycelial samples of L. gibbosa with sawdust and non-sawdust treatments. The transcripts were compared and analyzed using such as eggNOG annotations, GO annotations, and KEGG annotations to predict and screen genes associated with wood degradation. Fluorescence quantitative PCR (qPCR) was used to detect the expression quantities of eleven genes such as mnp2 related to lignin degradation under 5 days of sawdust treatment, and the transcriptome data were used to analyze the genes' expression levels.[Result] A total of 38.9 Gb clean data were obtained from the transcriptome sequencing of L. gibbosa. The average clean data of each sample reach 6.49 Gb. The mapped ratio between the reads of each sample and the reference genome ranged from 71.23% to 74.25%. The edgeR software was used to analyze the genes' differential expression, and the results showed that 898 differentially expressed genes (DEGs) were obtained, among which 351 genes were up-regulated and 547 genes down-regulated. There were 251 genes annotated by GO database, 223 genes by KEGG database, and 704 genes by eggNOG database. eggNOG analysis showed that the expressions of DEGs were mostly clustered under the functional classifications of energy production and conversion, post-transcriptional modification, protein metabolism, chaperone relationship, biosynthesis of secondary metabolites, carbohydrate transport and catabolism. GO analysis showed that the significantly enriched biological processes with higher frequency were pyruvate metabolic process, isoprenoid biosynthetic process, aspartate family amino acid metabolic process and lignin catabolic process. In KEGG pathway analysis, the DEGs were distributed in 86 different biological pathways, and the major metabolic pathways with significantly enriched DEGs were carbon metabolism, citrate cycle, degradation of aromatic compounds, and glyoxylate metabolism. The result of qPCR showed that under the sawdust treatment the expressions of mnp2, mnp3, lip9, lip2, laccase1, and nadp were significantly up-regulated, and the expressions of mnp10s, mrp, cyp450-1, GroES1, and GroES2 were significantly down-regulated. qPCR and transcriptome sequencing showed that there was slight deviation in the difference multiples of expression, but the overall trend was consistent, which proved that transcriptome sequencing result were correct and reliable.[Conclusion] The wood degradation by L. gibbosa of is closely related to the pathways of carbon metabolism, degradation of aromatic compounds, and other biological processes such as lignin catabolic process. According to the result of genes functional annotation, 11 important differentially expressed genes related to the lignin degradation by white rot fungi were obtained.

Key words: Lenzites gibbosa, white rot fungi, transcriptome, lignin, degradation

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