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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2020, Vol. 46 Issue (5): 571-581    DOI: 10.3785/j.issn.1008-9209.2020.02.012
植物保护     
照光抑制稻曲病菌菌丝生长的转录组分析
郭宇燕(),张璐,赵心雨,胡东维,梁五生()
浙江大学农业与生物技术学院生物技术研究所/农业部作物病虫分子生物学重点实验室，杭州 310058
Transcriptomic analysis of the lighting inhibition on growth of Villosiclava virens mycelia
Yuyan GUO(),Lu ZHANG,Xinyu ZHAO,Dongwei HU,Wusheng LIANG()
Institute of Biotechnology, College of Agriculture and Biotechnology/Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Zhejiang University, Hangzhou 310058, China
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摘要：

为探究稻曲病菌（Villosiclava virens）菌丝生长的调控因子及其调控机制，将稻曲病菌ZJ09菌株用马铃薯蔗糖琼脂（potato sucrose agar, PSA）平板培养基分别于黑暗和照光（日光灯提供的白光）条件下培养。结果显示：照光能显著抑制菌丝生长，说明光照条件是该菌菌丝生长的一种调控因子。采集上述2组菌丝进行转录组测序，共获得695个差异表达基因（differentially expressed genes, DEGs），其中359个基因在照光后表达上调，336个基因在照光后表达下调。从DEGs中选取6个基因用实时荧光定量聚合酶链式反应（real-time quantitative polymerase chain reaction, RT-qPCR）进行验证，所得结果与转录组测序结果一致。对DEGs进行基因本体（gene ontology, GO）富集分析，结果491个DEGs得到富集，涉及生物过程、细胞成分和分子功能3大类别，说明照光抑制菌丝生长的机制较为复杂。对DEGs进行京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路注释，结果表明：照光抑制菌丝生长主要与核糖体结构和功能，以及氨基酸代谢受到影响有关。DEGs包含1个乙酰辅酶A合成酶编码基因，照光显著抑制该基因表达，据此推测，削弱菌丝的乙酰辅酶A合成能力可能是照光抑制菌丝生长的机制之一。由于UvHOG1和UvBI-1这2个基因都不是DEG，因此蛋白激酶HOG1（high osmolarity glycerol 1）和Bax抑制子1可能与照光对菌丝生长的抑制作用无关。
关键词： 稻曲病菌;  照光;  转录组;  差异表达基因    
Abstract:

With the aim of exploring the regulating factors and involved mechanisms for the growth of Villosiclava virens mycelia, a strain (ZJ09) of this fungus was cultured on potato sucrose agar (PSA) plates under dark and lighting (with white light from fluorescents) conditions, respectively, and the growth states of the colonies were compared. A significant inhibitory effect was observed by lighting on the growth of V. virens mycelia, which suggested that lighting condition was a regulating factor for the mycelial growth of this fungus. The mycelial samples of the fungal strain ZJ09 from the dark and lighting groups were collected and their transcriptomes were sequenced, respectively. Six hundred and ninety-five differentially expressed genes (DEGs) caused by lighting were identified, including 359 up-regulated genes and 336 down-regulated ones. Six DEGs were selected out to check by real-time quantitative polymerase chain reaction (RT-qPCR) and the expression results were consistent with those by transcriptomic sequencing. During gene ontology (GO) enrichment analysis, 491 of the 695 DEGs were enriched to the terms of biological process, cellular component and molecular function, indicating that complex mechanisms may be involved in the inhibitory effect of lighting on mycelial growth. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis results of the 695 DEGs indicated that influences on the structure and functions of ribosome, and amino acid metabolism were quite probably involved in the inhibitory effect of lighting on mycelial growth. As DEGs included a gene encoding acetyl-coenzyme A synthetase and the lighting condition significantly inhibited its expression, it was inferred that impairing the mycelial acetyl-coenzyme A synthesis ability was one of mechanisms of the inhibitory effect of lighting on mycelial growth. In contrast, as neither of the genes UvHOG1 and UvBI-1 was a DEG, the protein kinase HOG1 (high osmolarity glycerol 1) and Bax inhibitor-1 may both have nothing to do with the inhibitory effect of lighting on mycelial growth.
Key words: Villosiclava virens    lighting    transcriptome    differentially expressed gene
收稿日期: 2020-02-01 出版日期: 2020-11-19
CLC:  S 435.111.46  
基金资助: 浙江省自然科学基金(LY20C140007);国家自然科学基金(31671969)
通讯作者: 梁五生     E-mail: guoyuyan@zju.edu.cn;liangws@zju.edu.cn
作者简介: 郭宇燕（https://orcid.org/0000-0002-7975-4635），E-mail：guoyuyan@zju.edu.cn
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引用本文:

郭宇燕,张璐,赵心雨,胡东维,梁五生. 照光抑制稻曲病菌菌丝生长的转录组分析[J]. 浙江大学学报（农业与生命科学版）, 2020, 46(5): 571-581.

Yuyan GUO,Lu ZHANG,Xinyu ZHAO,Dongwei HU,Wusheng LIANG. Transcriptomic analysis of the lighting inhibition on growth of Villosiclava virens mycelia. Journal of Zhejiang University (Agriculture and Life Sciences), 2020, 46(5): 571-581.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2020.02.012    或    http://www.zjujournals.com/agr/CN/Y2020/V46/I5/571

序号

No.

基因编号

Gene ID

基因名称

Gene name

引物序列（5′→3′）

Primer sequence (5′→3′)

1UVI_02009420

α微管蛋白-1

α-tubulin-1

F: AGGTTGCGTTGAAGGAGGTT

R: GAGGTGGAGTTGCCGATAAA

2UVI_02055420

二氢硫辛酰胺支链转酰酶

Dihydrolipoamide branched chain transacylase

F: TGTGGTGCTCTTCGGTCTTC

R: GATCCTCTCGGAAACCAGCC

3UVI_02000470

4-羟基苯丙酮酸双加氧酶

4-hydroxyphenylpyruvate dioxygenase

F: CATTGCCCTCCACTTCAGCA

R: CAAAGGCAAACGTGGTGGTG

4UVI_02002140

尿黑酸1，2-二氧酶

Homogentisate 1, 2-dioxygenase

F: GCTCTGCTTGTTTTCGTGCC

R: GGAAGCTGTTCAAGGAGCGT

5UVI_02004190

推定的1，3（4）-β-葡聚糖内切酶

Putative endo-1, 3 (4)-beta-glucanase

F: CGCACCGACGACAATCAA

R: TTGGGACCGAGGGAAGAA

6UVI_02026880

膜整合蛋白

Integral membrane protein

F: AGAAGGCAAGTCACAGCCAA

R: ACCCCGTAGTGCATCAATCG

7UVI_02021740

细胞分裂控制蛋白12

Cell division control protein 12

F: CCTGTCCGACCCCTCG

R: CAGCTGGTGCTGGCTGAC
表1  实时荧光定量PCR试验所用引物
图1  照光对PSA平板上ZJ09菌株菌落生长的影响A.菌落正面；B.菌落背面；C.菌落直径。**表示在P＜0.01水平差异有高度统计学意义。
图2  黑暗组与照光组ZJ09菌株菌落状态的比较ZJ09菌株在PSA平板上培养20 d后拍照，菌落内部图片放大倍数为100，菌落边缘图片放大倍数为208。

文库

Library

高质量识别序列

Clean reads

高质量识别序列碱基

Base of clean reads

Q30

模糊碱基N占比

Percentage of

ambiguous base N/%

数量

Number

占比

Percentage/%

数量

Number

占比

Percentage/%

CK_D143 496 29298.826 454 991 96097.776 142 237 0310.001 710
CK_D241 555 41298.716 168 929 66297.695 866 096 2100.001 647
Uv_L144 525 23497.916 639 237 02297.336 358 678 5890.002 933
Uv_L245 389 29298.786 731 516 25497.666 414 065 1090.001 662
表2  转录组输出数据及质量的基本情况

样品

Sample

高质量识别

序列总数

Total number

of clean reads

比对参考基因组的高质量识别序列

Clean reads mapped to

the reference genome

比对到多个位置的

高质量识别序列

Multiply-mapped clean reads

仅比对到一个位置的

高质量识别序列

Uniquely-mapped clean reads

数量

Number

占比

Percentage/%

数量

Number

占比

Percentage/%

数量

Number

占比

Percentage/%

CK_D143 496 29237 712 53686.70600 6821.5937 111 85498.41
CK_D241 555 41235 740 54186.01532 4601.4935 208 08198.51
Uv_L144 525 23438 937 98087.451 100 5602.8337 837 42097.17
Uv_L245 389 29238 928 19485.77626 4001.6138 301 79498.39
表3  过滤后的测序数据与参考基因组序列比对结果
图3  稻曲病菌菌丝差异表达基因的GO富集分析结果

通路注释

Description of pathway

通路ID

Pathway ID

P值

P value

差异表达基因数目

Number of DEGs

核糖体 Ribosomeko03010＜0.000 00159
缬氨酸、亮氨酸和异亮氨酸降解 Valine, leucine and isoleucine degradationko002800.000 006 9711
酪氨酸代谢 Tyrosine metabolismko003500.000 074 78
赖氨酸生物合成 Lysine biosynthesisko003000.000 7375
谷胱甘肽代谢 Glutathione metabolismko004800.002 277
β-丙氨酸代谢 Beta-alanine metabolismko004100.004 016
半胱氨酸和蛋氨酸代谢 Cysteine and methionine metabolismko002700.004 329
苯丙氨酸代谢 Phenylalanine metabolismko003600.005 826
丙酸盐代谢 Propanoate metabolismko006400.008 176
药物代谢-细胞色素P450 Drug metabolism-cytochrome P450ko009820.011 93
细胞色素P450对异种生物的代谢 Metabolism of xenobiotics by cytochrome P450ko009800.011 93
硒复合代谢 Selenocompound metabolismko004500.011 93
丙酮酸代谢 Pyruvate metabolismko006200.017 17
碳水化合物消化吸收 Carbohydrate digestion and absorptionko049730.021 83
赖氨酸降解 Lysine degradationko003100.043 75
苯乙烯降解 Styrene degradationko006430.051 53
泛醌和其他萜类醌生物合成 Ubiquinone and other terpenoid-quinone biosynthesisko001300.051 53
丁酸盐代谢 Butanoate metabolismko006500.063 44
酮体的合成与降解 Synthesis and degradation of ketone bodiesko000720.065 02
氰基氨基酸代谢 Cyanoamino acid metabolismko004600.071 03
表4  稻曲病菌菌丝差异表达基因的KEGG通路分析结果
图4  转录组数据的RT-qPCR验证编号1～6分别对应表1中编号2～7所代表的基因。FC：差异倍数。转录组测序的FC=（Uv_L1和Uv_L2的平均值）/（CK_D1和CK_D2的平均值）。RT-qPCR的FC值用2－△△CT法计算，所示结果为3次试验的平均值。
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