双孢蘑菇转录组测序及褐变相关基因的挖掘
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摘要
为从分子水平研究双孢蘑菇多酚氧化酶调控褐变的机理,以采后贮藏第1、7、14天的双孢蘑菇菌盖为材料,应用转录组测序技术对双孢蘑菇菌盖RNA进行测序分析。共获得168 607 212个高质量的clean reads;贮藏第7天和第1天相比筛选出727个差异表达基因(differentially expressed genes,DEGs),贮藏第14天和第1天相比筛选出1 524个DEGs;Gene Ontology(GO)功能富集和Pathway富集分析将这些差异表达基因归类于180个代谢途径;从已获得的差异表达基因中筛选得到PPO1、PPO3、PPO5、LAC1和LAC4五个多酚氧化酶相关基因。研究结果为双孢蘑菇功能相关基因挖掘和品质改良提供理论数据支撑。
In order to study how polyphenol oxidase regulates the progression of Agaricus bisporus browning, we performed mRNA sequencing using the RNA-seq technique on the pilei of A. bisporus collected on day 1, 7 and 14 of postharvest storage. A total of 168 607 212 high-quality reads were obtained; 727 and 1 524 differentially expressed genes were identified on day 7 and 14 compared with day 1 of storage, respectively. These differentially expressed genes were classified into 180 metabolic pathways after gene ontology and pathway enrichment analysis; of them, five polyphenol oxidase genes (PPO1, PPO3, PPO5, LAC1 and LAC4) were screened out and their relationships with browning were investigated. These results provide a theoretical basis for mining of functional genes and quality improvement of A. bisporus.
In order to study how polyphenol oxidase regulates the progression of Agaricus bisporus browning, we performed mRNA sequencing using the RNA-seq technique on the pilei of A. bisporus collected on day 1, 7 and 14 of postharvest storage. A total of 168 607 212 high-quality reads were obtained; 727 and 1 524 differentially expressed genes were identified on day 7 and 14 compared with day 1 of storage, respectively. These differentially expressed genes were classified into 180 metabolic pathways after gene ontology and pathway enrichment analysis; of them, five polyphenol oxidase genes (PPO1, PPO3, PPO5, LAC1 and LAC4) were screened out and their relationships with browning were investigated. These results provide a theoretical basis for mining of functional genes and quality improvement of A. bisporus.
引文
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[19]吴小梅,张昕,李南羿.双孢蘑菇子实体不同发育时期的转录组分析[J].菌物学报,2017,36(2):193-203.DOI:10.13346/j.mycosystema.150275.
[20]LEI J,LI B J,ZHANG N,et al.Effects of UV-C treatment on browning and the expression of polyphenol oxidase (PPO) genes in different tissues of Agaricus bisporus during cold storage[J].Postharvest Biology&Technology,2018,139:99-105.DOI:10.1016/j.postharvbio.2017.11.022.
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[24]MDELOSA S,ROZENFELD P A,MARTINEZ G A,et al.Polyphenoloxidase activity from strawberry fruit(Fragaria ananassa,Duch.cv Selva):characterisation and partial purification[J].Journal of the Science of Food&Agriculture,2010,80(9):1421-1427.DOI:10.1002/1097-0010(200007)80:9<1421::AID-JSFA649>3.0.CO;2-K.
[25]吴光美,严俊杰,杨志云,等.与金针菇子实体发育有关的漆酶编码基因Fv-lac4的结构及表达差异分析[J].基因组学与应用生物学,2014,33(6):1254-1260.DOI:10.13417/j.gab.033.001254.
[26]卓睿,马富英,周帅,等.糙皮侧耳生长发育过程中漆酶基因家族的表达研究[J].菌物学报,2015,34(4):712-716.DOI:10.13346/j.mycosystema.150075.
[27]VAN L J,WICHERS H J.Tyrosinase activity and isoform composition in seperate tissues during development of Agaricus bisporus fruit bodies[J].Mycological Research,1999,103:413-418.DOI:10.1017/s095375629800731x.
[2]LI N Y,CAI W M,JIN Q L,et al.Molecular cloning and expression of polyphenol oxidase genes from the mushroom Agaricus bisporus[J].Agriculture Sciences in China,2011,10(2):185-194.DOI:10.1016/s1671-2927(09)60305-9.
[3]WU X L,GUAN W Q,YAN R X,et al.Effects of UV-C on antioxidant activity,total phenolics and main phenolic compounds of the melanin biosynthesis pathway in different tissues of button mushroom[J].Postharvest Biology and Technology,2016,118:51-58.DOI:10.1016/j.postharvbio.2016.03.017.
[4]CHENG Y D,LIU L Q,ZHAO G Q,et al.The effects of modified atmosphere packaging on core browning and the expression patterns of PPO and PAL genes in‘Yali’pears during cold storage[J].LWT-Food Science and Technology,2015,60(2):1243-1248.DOI:10.1016/j.lwt.2014.09.005.
[5]张煜晗,张希,董莉,等.草莓多酚氧化酶基因全长的克隆及表达载体构建[J].北京农学院学报,2018,33(2):7-14.
[6]张慧,田新权,高巍,等.陆地棉P P O基因全基因组鉴定及对黄萎病菌的响应分析[J].棉花学报,2017,29(5):428-436.DOI:10.11963/1002-7807.zhll.20170727.
[7]CHI M,BHAGWAT B,LANE W D,et al.Reduced polyphenol oxidase gene expression and enzymatic browning in potato (Solanum tuberosum L.) with artificial microRNAs[J].BMC Plant Biology,2014,14:62.DOI:10.1186/1471-2229-14-62.
[8]WICHERS H J,RECOURT K,HENDRIKS M,et al.Cloning,expression and characterisation of two tyrosinase cDNAs from Agaricus bisporus[J].Applied Microbiology&Biotechnology,2003,61(4):336-341.DOI:10.1007/s00253-002-1194-2.
[9]WEIJN A,BASTIAAN N S,WICHERS H J,et al.Melanin biosynthesis pathway in Agaricus bisporus mushrooms[J].Fungal Genetics&Biology,2013,55(6):42-53.DOI:10.1016/j.fgb.2012.10.004.
[10]冉欣欣.双孢蘑菇PPO基因家族的鉴定和部分基因功能分析[D].杭州:浙江农林大学,2013:18-23.
[11]周华,张新,刘腾云,等.高通量转录组测序的数据分析与基因发掘[J].江西科学,2012,30(5):607-611.DOI:10.13990/j.issn1001-3679.2012.05.035.
[12]许波,张伟强,冯晓曦,等.转录组测序技术在玉米中的应用研究进展[J].玉米科学,2014,22(1):67-72;78.DOI:10.13597/j.cnki.maize.science.2014.01.014.
[13]QI Y X,LIU Y B,RONG W H.RNA-Seq and its applications:a new technology for transcriptomics[J].Hereditas (Beijing),2011,33(11):1191-1202.DOI:10.3724/sp.j.1005.2011.01191.
[14]贾昌路,张瑶,朱玲,等.转录组测序技术在生物测序中的应用研究进展[J].分子植物育种,2015,13(10):2388-2394.DOI:10.13271/j.mpb.013.002388.
[15]石浩然.基于二代测序的转录组数据分析方法的比较研究[D].雅安:四川农业大学,2016:1-5.
[16]姚娜,刘秀明,董园园,等.转录组的测序方法及应用研究概述[J].北方园艺,2017(12):192-198.DOI:10.11937/bfyy.201712043.
[17]TANG L H,JIAN H H,SONG C Y,et al.Transcriptome analysis of candidate genes and signaling pathways associated with light-induced brown film formationin Lentinula edodes[J].Applied Microbiology and Biotechnology,2013,97(11):4977-4989.DOI:10.1007/s00253-013-4832-y.
[18]易琳琳.香菇转录组测序及细胞壁降解相关基因筛选的研究[D].杭州:浙江大学,2015:25-38.
[19]吴小梅,张昕,李南羿.双孢蘑菇子实体不同发育时期的转录组分析[J].菌物学报,2017,36(2):193-203.DOI:10.13346/j.mycosystema.150275.
[20]LEI J,LI B J,ZHANG N,et al.Effects of UV-C treatment on browning and the expression of polyphenol oxidase (PPO) genes in different tissues of Agaricus bisporus during cold storage[J].Postharvest Biology&Technology,2018,139:99-105.DOI:10.1016/j.postharvbio.2017.11.022.
[21]SHU C,SUN S,CHEN J,et al.Comparison of different methods for total RNA extraction from sclerotia of Rhizoctonia solani[J].Electronic Journal of Biotechnology,2014,17(1):50-54.DOI:10.1016/j.ejbt.2013.12.009.
[22]YOUNG M D,WAKEFIELD M J,SMYTH G K,et al.Gene ontology analysis for RNA-seq:accounting for selection bias[J].Genome Biology,2010,11(2):R14.DOI:10.1186/gb-2010-11-2-r14.
[23]KANEHISA M,ARAKI M,GOTO S,et al.KEGG for linking genomes to life and the environment[J].Nucleic Acids Research,2008,36(Database issuse):408-410.DOI:10.1093/nar/gkm882.
[24]MDELOSA S,ROZENFELD P A,MARTINEZ G A,et al.Polyphenoloxidase activity from strawberry fruit(Fragaria ananassa,Duch.cv Selva):characterisation and partial purification[J].Journal of the Science of Food&Agriculture,2010,80(9):1421-1427.DOI:10.1002/1097-0010(200007)80:9<1421::AID-JSFA649>3.0.CO;2-K.
[25]吴光美,严俊杰,杨志云,等.与金针菇子实体发育有关的漆酶编码基因Fv-lac4的结构及表达差异分析[J].基因组学与应用生物学,2014,33(6):1254-1260.DOI:10.13417/j.gab.033.001254.
[26]卓睿,马富英,周帅,等.糙皮侧耳生长发育过程中漆酶基因家族的表达研究[J].菌物学报,2015,34(4):712-716.DOI:10.13346/j.mycosystema.150075.
[27]VAN L J,WICHERS H J.Tyrosinase activity and isoform composition in seperate tissues during development of Agaricus bisporus fruit bodies[J].Mycological Research,1999,103:413-418.DOI:10.1017/s095375629800731x.