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上海交通大学学报(农业科学版)  2016, Vol. 34 Issue (1): 74-80    DOI: 10.3969/J.ISSN.1671-9964.2016.01.013
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灰树花菌丝体与原基转录组差异表达分析
王伟科1(), 周祖法1, 陈青2, 袁卫东1()
1. 杭州市农业科学研究院, 杭州310024
2. 浙江省农业技术推广中心, 杭州310020
Analysis of Differentially Expressed Genes between Mycelium and Primordium of Grifola frondosa
Wei-ke WANG1(), Zu-fa ZHOU1, Qing CHEN2, Wei-dong YUAN1,*()
1.Hangzhou Academy of Agricultural Sciences, Hangzhou 310024, China
2.Zhejiang Agro-Tech Extension Center, Hangzhou 310020, China
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摘要 

为了解灰树花原基形成及子实体分化机制, 采用Illumina测序技术对灰树花菌丝体和原基进行了全转录组测序和数据分析, 对菌丝体和原基中的差异表达基因进行了研究。结果表明, 在菌丝体和原基中分别得到35788532个和32755254个高质量测序标签。差异表达基因分析表明, 两个文库中共有显著性差异表达的基因4094个, 其中在原基中上调、下调的基因数分别为1886和2208个, 只在原基中表达的基因284个。经Blastnr 比对, 在菌丝体与原基阶段差异表达的基因主要与酸性蛋白酶类、凝集素、细胞色素、NADPH-P450还原酶、酯酶、胺氧化酶、克拉维胺合成酶、糖苷水解酶家族相关。在原基中特异表达的基因主要与糖代谢、脂类代谢、核酸代谢及细胞膜、叶绿体膜有关。GO功能富集分析结果表明, 线粒体膜相关基因、谷氨酰胺代谢、脂肪酸生物合成相关基因均上调表达; Pathway 功能富集分析结果表明, 合成核糖体蛋白的基因均上调表达, 表明原基形成时细胞代谢增强, 蛋白质合成量增加。
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王伟科
周祖法
陈青
袁卫东
关键词 ： 灰树花,  菌丝体,  原基,  差异表达基因    
Abstract：

The expression profiling of mycelium and primordium of Grifola frondosa was analyzed with high-throughput sequencing technology based on the Illumina Genome Analyzer platform.Totally 35788532 and 32755254 clean reads from mycelium and primordium libraries were obtained.Analysis of differentially expressed genes shows that there are 4094 genes differentially expressed between two libraries, and in the primordium library, 1886 of them are up-regulated while 2208 of them are down-regulated.There are 284 genes that are only expressed in the primordium library.With Blastnr comparison, genes differently expressed between two libraries are found to be related to acid protease and lectin, cytochrome, NADPH-P450 reductase, esterase, amine oxidase, clavaminate synthase, glycoside hydrolase.Genes only expressed in the primordium are found to be related to glucose, lipid, nucleic acids metabolism and cell and chloroplast membrane structure.Gene ontology functional enrichment analysis reveals that most genes related to mitochondrial membrane, glutamine family amino acid metabolic process, fatty acid biosynthetic process are up-regulated.Pathway enrichment analysis shows that genes related to ribosome are all up-regulated, and this indicates that when primordium is developed the cell metabolism speeds up and the protein synthesis increases.
Key words： Grifola frondosa    mycelium    primordium    differentially expressed gene
收稿日期: 2015-04-23      出版日期: 2016-03-08
基金资助:浙江省农业新品种选育重大科技专项部分内容(2012C12911)
作者简介:

作者简介: 王伟科(1981-), 男, 硕士, 高级农艺师, 研究方向:食药用菌育种与栽培技术, E-mail:akeok@126.com;

通讯作者: 袁卫东(1969-), 男, 高级农艺师, 研究方向:食药用菌育种与栽培技术, E-mail:ywd0507@126.com
引用本文:   
王伟科, 周祖法, 陈青, 袁卫东. 灰树花菌丝体与原基转录组差异表达分析[J]. 上海交通大学学报(农业科学版), 2016, 34(1): 74-80.
Wei-ke WANG, Zu-fa ZHOU, Qing CHEN, Wei-dong YUAN. Analysis of Differentially Expressed Genes between Mycelium and Primordium of Grifola frondosa. Journal of Shanghai Jiaotong University (Agricultural Sciences), 2016, 34(1): 74-80.
链接本文:  
http://www.qk.sjtu.edu.cn/jsjtua/CN/10.3969/J.ISSN.1671-9964.2016.01.013      或      http://www.qk.sjtu.edu.cn/jsjtua/CN/Y2016/V34/I1/74
样品
Sampels		 浓度
Concentration		 单位
Unit		 OD260/280 OD260/230
1-JST 266.4 ng·μL-1 2.14 1.92
2-YJ 697.8 ng·μL-1 2.16 2.21
Tab.1  灰树花菌丝体、原基RNA浓度
Fig.1  灰树花菌丝体、原基RNA电泳检测

1-JST表示菌丝体, 2-YJ表示原基

数据产出及比对
Data output and comparison		 菌丝体
Mycelium		 原基
Primordium
原始序列标签
Rawreads		 35913116 32873980
高质量测序标签
Cleanreads		 35788532 32755254
未比对上的标签
Unmapped reads		 9028139 7574132
非唯一比对位置标签
Mapq<mapq_cut(non-unique)		 4665894 3623751
唯一比对位置标签
Mapq>=apq_cut(unique)		 22085081 21547780
第一端测序标签
Read-1		 11010040 10756337
第二端测序标签
Read-2		 11075041 10791443
比对到基因组上正列
Reads map to‘+’		 10981873 10704107
比对到基因组上负列
Reads map to‘-’		 11103208 10843673
整段比对到外显子的序列
Non-splice reads		 22061957 21537230
分段比对到两个外显子上的序列
Splice reads		 23124 10550
双端测序序列定位到基因组上相对
的距离符合测序片段的长度分布
Eads mapped in proper pairs		 19597840 19326088
Tab.2  两个样本中标签分布一览表
基因ID
Gene ID		 菌丝中表达值
Expression value of
mycelium		 原基中表达值
Expression value of
primordium		 Q值
Q value		 同源基因及其功能
Homologous gene and its function
unigene23873 483.4 6.0 0.023 酸性蛋白酶(gi|392558359|)(gb|EIW51548.1|)
Acid protease
unigene13929 11.6 5860.5 0.037 凝集素(gi|18478668|)(gb|AAL73235.1|)
unigene30943 186.4 1.4 0.037 DUF1479结构域蛋白(gi|392564554|)(gb|EIW57732.1|)
DUF1479 domain containing protein
unigene12210 8.2 2412.5 0.042 凝集素(gi|50980806|)(gb|AAT91249.1|)
lectin
unigene41873 2.3 81.1 0.042 细胞色素P450(gi|395327404|)(gb|EJF59804.1|)
Cytochrome P450
unigene45937 1.7 67.0 0.042 NADPH-P450还原酶(gi|392559250|)(gb|EIW52435.1|)
NADPH-P450 reductase
unigene27346 223.5 5.9 0.044 IlvN结构域蛋白(gi|395334382|)(gb|EJF66758.1|)
IlvN domain containing protein
unigene28323 3.5 162.9 0.044 酯酶(gi|395328576|)(gb|EJF60967.1|)
esterase
unigene41874 2.1 68.9 0.044 细胞色素P450(gi|395327404|)(gb|EJF59804.1|)
Cytochrome P450
unigene45936 2.2 73.6 0.044 NADPH-P450还原酶(gi|392559250|)(gb|EIW52435.1|)
NADPH-P450 reductase
unigene41875 3.0 170.3 0.048 细胞色素P450(gi|395327404|)(gb|EJF59804.1|)
Cytochrome P450
unigene14635 3.5 118.4 0.052 真菌疏水蛋白结构(gi|390605204|)(gb|EIN14595.1|)
Fungal hydrophobin
unigene47574 248.0 1.3 0.056 胺氧化酶(gi|392563541|)(gb|EIW56720.1|)
Amine oxidase
unigene19870 22.8 653.1 0.058 真菌疏水蛋白结构(gi|395323171|)(gb|EJF55662.1|)
Fungal hydrophobin
unigene36089 1.8 34.7 0.058 细胞色素P450(gi|390599619|)(gb|EIN09015.1|)
Cytochrome P450
unigene30525 0.8 13.6 0.06 克拉维胺合成酶(gi|392569544|)(gb|EIW62717.1|)
Clavaminate synthase-like protein
unigene36088 1.5 27.1 0.06 细胞色素P450(gi|390599619|)(gb|EIN09015.1|)
Cytochrome P450
unigene39627 14.6 1.0 0.061 类CDF转运蛋白(gi|395334914|)(gb|EJF67290.1|)
CDF-like metal transporter
unigene30523 0.5 14.7 0.063 克拉维胺合成酶(gi|392569544|)(gb|EIW62717.1|)
Clavaminate synthase-like protein
unigene2984 69.6 0.3 0.065 糖苷水解酶家族(gi|392564918|)(gb|EIW58095.1|)
Glycoside hydrolase family protein
Tab.3  菌丝体、原基中差异表达基因的同源比对
同源基因功能
Functions of
homologous genes		 同源基因Homologous gene
糖代谢相关
Glucose metabolism		 E1脱氢酶及转酮醇酶结构域蛋白(gi|395325950|)(gb|EJF58365.1|),
dehydrogenase E1 and transketolase domain-containing protein
醛脱氢酶(gi|392561496|)(gb|EIW54677.1|),
Aldehyde dehydrogenase
MFS通用底物转运蛋白(gi|392561630|)(gb|EIW54811.1|)
MFS general substrate transporter
脂类代谢相关
Lipid metabolism		 酮脂酰合成酶结构域蛋白(gi|395326786|)(gb|EJF59192.1|)
ketoacyl-synt-domain-containing protein
核酸代谢相关
Nucleotide etabolism		 HAD水解酶(gi|395328072|)(gb|EJF60467.1|)
HAD hydrolase
细胞膜蛋白
Cell membrane protein		 DUF6结构域蛋白(gi|392559753|)(gb|EIW52937.1|),
DUF6-domain-containing protein
NAD(P)结合蛋白(gi|392588861|)(gb|EIW78192.1|)
NAD(P)-binding protein
叶绿体膜蛋白
Chloroplast membrane protein		 磷酸丙糖转运蛋白家族(emb|AJ437264.1)
phosphate translocator-like protein
其他
Others		 细胞色素(gi|395325039|)(gb|EJF57468.1|),
cytochrome
叶绿素A-B 结合蛋白PF00504(gb|EF576336.1|),
Chlorophyll A-B binding protein PF00504
类克拉维胺合成酶(gi|395330466|)(gb|EJF62849.1|),
Clavaminate synthase-like protein
乌头酸水合酶(gi|4029334|)(emb|CAA76360.1|)
Aconitate hydratase
Tab.4  原基中特异表达基因的同源比对
基因本体条目
Gene ontology entries		 分类
Classification		 P值
P value		 上调基因数
Up-regulated genes		 下调基因数
Down-regulated genes
结合ATP
ATP binding		 分子功能
Molecular function		 9.9E-06 74 30
酸酐水解酶活性
Hydrolase activity		 分子功能
Molecular function		 2.1E-07 46 5
胞内成分
Intracellular part		 细胞组成
Cellular component		 3.1E-07 91 24
结合过渡金属离子
Transition metal ion binding		 分子功能
Molecular function		 9.8E-09 102 52
线粒体膜成分
Mitochondrial membrane part		 细胞组成
Cellular component		 9.9E-08 6 0
结合金属离子
Metal ion binding		 分子功能
Molecular function		 1.6E-11 117 60
结合核酸
Nucleic acid binding		 分子功能
Molecular function		 8.0E-13 115 24
谷氨酰胺家族氨基酸代谢过程
Glutamine family amino acid metabolic process		 生物过程
Biological process		 1.4E-05 8 0
肽酶活性
Peptidase activity		 分子功能
Molecular function		 6.6E-11 27 25
结合阳离子
Cation binding		 分子功能
Molecular function		 2.9E-11 117 60
脂肪酸生物合成过程
Fatty acid biosynthetic process		 生物过程
Biological process		 4.8E-06 4 0
单糖分解代谢过程
Monosaccharide catabolic process		 生物过程
Biological process		 7.7E-06 2 5
胞内蛋白代谢过程
Cellular protein metabolic process		 生物过程
Biological process		 2.2E-06 41 2
小分子代谢过程
Small molecule metabolic process		 生物过程
Biological process		 6.7E-06 21 10
金属内肽酶活性
Metalloendopeptidase activity		 分子功能
Molecular function		 1.1E-06 9 1
水解酶活性
Hydrolase activity		 分子功能
Molecular function		 1.5E-09 120 62
结合嘌呤核苷三磷酸
Purine ribonucleoside triphosphate binding		 分子功能
Molecular function		 3.2E-07 95 32
氧化还原酶活性
Oxidoreductase activity		 分子功能
Molecular function		 1.1E-16 141 59
依赖NTP嘌呤解旋酶活性
Purine NTP-dependent helicase activity		 分子功能
Molecular function		 2.6E-07 17 3
单价无机阳离子跨膜运输活性
Monovalent inorganic cation transmembrane
transporter activity		 分子功能
Molecular function		 1.1E-05 6 5
Tab.5  菌丝体与原基GO功能富集分析
代谢途径
Metabolic pathway		 P值
P value		 上调基因数
Up-regulated genes		 下调基因数
Downk-regulated genes
氧化磷酸化途径
Oxidative phosphorylation		 7.2E-05 12 1
萜类生物合成途径
Terpenoid backbone biosynthesis		 0.000118 6 0
核糖体
Ribosome		 1.8E-06 21 0
花生四烯酸代谢途径
Arachidonic acid metabolism		 6.0E-05 4 1
莨菪、哌啶、吡啶类生物碱合成
Tropane, piperidine and pyridine alkaloid biosynthesis		 5.7E-05 5 2
mRNA检测途径
mRNA surveillance pathway		 0.000155 14 0
次级代谢产物生物合成途径
Biosynthesis of secondary metabolites		 2.4E-07 50 15
色氨酸代谢
Tryptophan metabolism		 0.000225 14 5
丙氨酸、天冬酰胺、谷氨酸代谢相关
Alanine, aspartate and glutamate metabolism		 0.000194 6 1
脂肪酸代谢相关
Fatty acid metabolism		 3.4E-05 13 1
苯丙氨酸、酪氨酸、色氨酸生物合成途径
Phenylalanine, tyrosine and tryptophan biosynthesis		 0.000238 8 0
Tab.6  菌丝体与原基pathway 显着富集分析列表
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