水杨酸诱导下蝙蝠蛾拟青霉转录组分析及虫草酸代谢途径关键酶基因挖掘

Transcriptome analysis of Samsoniella hepialiinduced by salicylic acid and crucial genes digging for metabolic pathways of cordycepic acid

摘要:

【目的】虫草酸是虫草中重要的活性成分之一，但其低含量极大地限制了其工业应用。水杨酸(salicylic acid, SA)是一种非生物诱导子，可以显著提高蝙蝠蛾拟青霉中虫草酸的合成，但蝙蝠蛾拟青霉虫草酸代谢途径及其对水杨酸的响应尚不明确。本研究旨在获得蝙蝠蛾拟青霉响应SA处理的转录组学信息，挖掘蝙蝠蛾拟青霉中虫草酸代谢途径关键酶基因。【方法】采用SA诱导培养蝙蝠蛾拟青霉，8 h后选取诱导和未诱导的菌丝进行转录组高通量测序分析。【结果】测序最终获得40.37 Gb的clean data，拼接得到20 317条unigene，平均长度为1 357.13 bp，功能注释共获得13 592条unigene。差异基因分析共筛选出差异基因2 574个，其中有1 135个上调，1 439个下调。KEGG富集分析表明，差异基因主要富集于细胞周期、减数分裂、半乳糖代谢、DNA复制、糖醇脂类生物合成、甘油脂类代谢等KEGG通路中。进一步分析得到与虫草酸代谢相关的基因13条，其中参与虫草酸生物合成的基因glk、gpi、gla、mpi、fbp、mtld在SA处理后表达量上调，而涉及虫草酸消耗的基因mdh在SA处理后表达量下调，qRT-PCR验证结果与RNA-Seq数据基本一致。【结论】对SA诱导后的蝙蝠蛾拟青霉转录组进行分析，获得虫草酸代谢途径相关的候选基因，SA能够提高虫草酸合成相关基因表达的同时抑制消耗虫草酸的基因表达，从而提高虫草酸生物合成量。本研究为SA调控蝙蝠拟青霉虫草酸生物合成途径相关基因研究提供了依据，也为后期开展虫草酸代谢调控机制研究奠定了基础。

关键词:蝙蝠蛾拟青霉;水杨酸;转录组;虫草酸;代谢途径

Abstract:

[Objective]Cordycepic acid is one of the most important active substances in Samsoniella hepiali,but the low content greatly restricts its industrial application.Salicylic acid (SA) is an abiotic inducer,and we found that it could increase the content of cordycepic acid in S.hepiali.However,the metabolic pathways of cordycepic acid and its response to SA are not clear.This study was designed to obtain the transcriptome information of S.hepiali in response to SA treatment,and to explore key enzyme genes for the metabolic pathways of cordycepic acid.[Methods] In this study,SA was used to induce S.hepiali,and after 8 h treatment,high-throughput transcriptome sequencing was performed on induced and uninduced mycelia.[Results] A total of 40.37 Gb clean data was acquired,and 20 317 unigenes with an average length of 1 357.13 bp were constructed.Additionally,13 592 unigenes were annotated in the public databases NR,NT,KEGG,Swissprot,GO,and Pfam,and 2 574 differentially expressed genes were identified,in which 1 135 were up-regulated and 1 439 were down-regulated.KEGG enrichment analysis demonstrated that the differentially expressed genes were concentrated in the pathways such as cell cycle,meiosis,galactose metabolism,DNA replication,glycolipid biosynthesis,and glyceride metabolism.Furthermore,13 unigenes involved in metabolic pathways of cordycepic acid were found based on the assignment of KEGG pathway.The expression of genes glk,gpi,gla,mpi,fbp and mtld related to cordycepic acid biosynthesis was all up-regulated after the induction of SA,yet that of the gene mdh involved in cordycepic acid consumption was down-regulated,and the qRT-PCR results were in substantial agreement with RNA-Seq data.[Conclusion] Transcriptome information of S.hepiali induced by SA was obtained,and candidate genes for the metabolic pathways of cordycepic acid were identified.SA improved the expression of the biosynthetic genes and inhibited the gene transcription related to cordycepsic acid consumption,thus increasing the accumulation of cordycepic acid.This study provides reference for regulating the genes related to the biosynthetic pathways of cordycepic acid in S.hepiali by SA,and also facilitates the investigation on the regulatory mechanism of cordycepic acid metabolism.

Key words:Samsoniella hepiali;salicylic acid;transcriptome;cordycepic acid;metabolic pathways