病毒感染海洋球石藻<i>Emiliania huxleyi</i>的转录组分析

田雪; 蔡伟聪; 苏金净; 吴书燕; 刘静雯

doi:10.3969/j.issn.0253-4193.2019.12.010

病毒感染海洋球石藻Emiliania huxleyi的转录组分析

doi: 10.3969/j.issn.0253-4193.2019.12.010

田雪^{1, 2,},
蔡伟聪^{1, 2},
苏金净^{1, 2},
吴书燕^{1, 2},
刘静雯^{1, 2, ,}

1.
集美大学食品与生物工程学院，福建厦门 361021
2.
福建省食品微生物与酶工程重点实验室，福建厦门 361021

基金项目: 国家自然科学基金项目（41576166）；福建省自然科学基金项目（2019J01696）。

详细信息

作者简介:
田雪（1992—），女，河南省南阳市人，主要从事海洋微生物生化与分子生物学研究。E-mail: yytx1216@126.com

通讯作者:
刘静雯，教授，主要从事海洋微型生物分子生物学研究。E-mail: ljwsbch@163.com;jwliu@jmu.edu.cn

中图分类号: Q78
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出版历程
- 收稿日期: 2018-12-19
- 修回日期: 2019-05-30
- 网络出版日期: 2021-04-21
- 刊出日期: 2019-12-25

Transcriptome analysis of marine microalga Emiliania huxleyi in response to virus infection

Tian Xue^{1, 2
,},
Cai Weicong^{1, 2},
Su Jinjing^{1, 2},
Wu Shuyan^{1, 2},
Liu Jingwen^{1, 2
, ,}

1.
Food and Bioengineering College of Jimei University, Xiamen 361021, China
2.
Key Laboratory of Food Microbiology and Enzyme Engineering of Fujian Province, Xiamen 361021, China

摘要

摘要: 海洋球石藻Emiliania huxleyi及其特异性裂解病毒E. huxleyi virus (EhVs)在调节海洋碳、硫循环及全球气候变化中起着重要作用，也是开展真核生物病毒–宿主相互作用研究的良好模型系统之一。为了探究病毒感染条件下E. huxleyi基因表达水平的变化，以海洋球石藻E. huxley–BOF 92及其专一性裂解病毒EhV-99B1为研究对象，利用Illumina HiSeq 2000高通量测序技术，分别对E. huxleyi病毒感染组(Exp)和非感染对照组(Con)6 h和45 h的藻细胞样品进行转录组测序分析。共得到32 909条平均长度为1 153 bp的基因。病毒感染6 h和45 h分别得到2 617和5 229个差异表达基因，其中共差异表达基因465个。随机选取10条差异表达基因，采用qRT-PCR进行实验验证，结果证实转录组分析可靠。GO功能注释和KEGG通路富集，发现大量基因与氧化应激反应、脂类代谢、碳水化合物代谢及信号转导等代谢过程相关，其中变化最显著的是谷胱甘肽代谢途径。从病毒感染球石藻转录组中筛选出部分与氧化应激反应相关的基因，其中9个基因显著上调，11个基因显著下调，表明宿主能够通过体内的氧化应激反应响应病毒胁迫。
- 海洋球石藻Emiliania huxleyi /
- 病毒感染 /
- 转录组 /
- 差异表达基因 /
- qRT-PCR /
- 氧化应激反应
Abstract: Emiliania huxleyi, the numerically dominant coccolithophore in the modern oceans and its specific lytic virus EhV exert a critical impact upon the oceanic carbon, sulfur cycle and global climate, thus serving as a key host-pathogen model system. Despite their impact on biogeochemical cycling, the transcriptional dynamics of these important oceanic events is still poorly understood. To understand the host-virus interaction in E. huxleyi-EhV system, the transcriptome of E. huxleyi BOF92 involved in virus infection was investigated by using Illumina HiSeq 2 000 high-throughput sequencing technology. Two cDNA libraries, generated 6 h and 45 h after viral infection (Exp) were compared with two libraries from the corresponding times uninfected cultures (Con). A total of 32 909 unigenes with an average length of 1 153 bp were generated. Totally 2 617 and 5 229 differentially expressed genes (DEGs) associated with viral infection were identified in 6 hpi and 45 hpi, respectively, among which 465 genes were the common DEGs in the two time points. Ten DEGs were random selected for quantitative RT-PCR (qRT-PCR) analysis, and the results confirmed that the transcriptome analysis was reliable. Furthermore, the DEGs were subject to GO and KEGG enrichment analysis. The results showed that most of the DEGs were involved in oxidation-reduction reactions, glutathione metabolism, lipid metabolism, carbohydrate metabolism and signal transduction. Some of the reactive oxygen species (ROS) scavenging genes were screened out, in which 9 genes were up-regulated and 11 genes were down-regulated. These results suggested that ROS signaling molecules might play a central role during host-virus interaction.
- Emiliania huxleyi /
- virus infection /
- transcriptome sequencing /
- differentially expressed genes /
- qRT-PCR /
- oxidative stress

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图 1 转录本的长度分布

Fig. 1 Length distribution of the transcripts

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图 2 病毒感染不同时间点差异表达基因的韦恩图

a. 上调差异基因韦恩图；b 下调差异基因韦恩图

Fig. 2 The Venn diagram of differentially expressed genes in 6 h and 45 h post infection

a. The number of up-regulation differentially expressed genes; b. the number of down-regulation differentially expressed genes

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图 3 10个差异表达基因的qRT-PCR验证

Fig. 3 Verification of ten differentially expressed genes by qRT-PCR

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图 4 差异表达基因GO富集结果

病毒感染6 h(a)和45 h(b)

Fig. 4 The most enriched GO terms and corresponding differentially expressed gene numbers of each term

Cells infected by the virus at 6 h (a) and 45 h (b)

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图 5 EhV感染重塑宿主谷胱甘肽代谢通路图

OPase：羟脯氨酸酶；MAP：膜氨肽酶I；GSH：γ-谷氨酰半胱氨酸合成酶；GGT：γ-谷氨酰转移酶；GSHS：谷胱甘肽合成酶；GR：胱甘肽还原酶；G6PDH：葡萄糖-6-磷酸脱氢酶；GPx：谷胱甘肽过氧化物酶；GDA：谷胱甘肽脱氢酶；SSase：亚精胺合成酶；ODase：鸟氨酸脱羧酶。基因名称框的颜色表示基因表达水平的倍数变化，log₂ FC > 0表示上调，log₂ FC < 0表示上调

Fig. 5 Glutathione metabolic pathways responding to virus infection

OPase: oxoprolinase; MAP: membrane aminopeptidase I; GSH: γ-glutamylcysteine synthetase; GGT: γ-glutamyl transferase; GSHS: glutathione synthetase; GR: glutathione reductase; G6PDH: glucose-6-phosphate dehydrogenase; GPx: glutathione peroxidase; GDA: glutathione dehydrogenase; SSase: spermidine synthase; ODase: ornithine decarboxylase. Color filling of gene name box depicts the fold change of expression levels of the gene based on RNA-seq data, log₂ FC > 0 indicating up- regulation, log₂ FC < 0 indicating down- regulation.

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表 1 实验所用的引物

Tab. 1 Primers used in the experiments

基因	正向引物序列 (5′-3′)	反向引物序列 (5′–3′)
β-Tublin (17257729)	TCATGTGCTCCTACTCGGTCTTC	TTCAGCGTGCGGAAACAGA
ATG13 (17261891)	GCGAAACTGCGTCCAGAAGA	CGCTCGAGAAGCACGAGATG
MC1 (17277722)	TTATCAGCGACGAGGACAGTTC	GCTCAACATGCCCTCCCTAG
MC2 (17251088)	TGGGGCTTTTCAGAGGAAGAT	CCGTCGCCTGAGTAAAAGATAA
MC3 (17269785)	CGACTGGCTGAATGAGGAGAA	ACCTTGTGGCTCTTGAGCATG
MC4 (17283241)	TCGCTGATGGTCTTTATGGA	TCCCTCGGAGGTCTCGTA
hSPT (17255778)	TCTCGGACACGCTCAACCA	CCCTCGACGATGATGATGATC
ATG3 (17287351)	TCAAGACAGTCACCCTCGAGTC	GGATGACGGAGGAGATGAACT
FADS (17253431)	CTTCTCCGAGATGCCCTTCTA	CAGTAGCCGAGAAACTCGGA
AMO (17252058)	GCTTGTGGTAACCTTCGTCC	GTGACCGCGTGAAACCAGT
DLD2 (19046437)	AATCATCGGGTCGGGGTAC	CATGGGGCTGGGCTACTAA

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表 2 测序数据量统计结果

Tab. 2 Statistics results of the sequencing data

样品	原始数据大小/bp	原始读段量	有效数据大小/bp	有效读段量	有效数据比率/%
Con_45_1	1 206 811 650	24 136 233	1 197 441 650	23 948 833	99.22
Con_45_2	1 206 812 550	241 362 51	1 197 176 200	23 943 524	99.20
Con_6_1	1 200 867 450	24 017 349	1 194 086 550	23 881 731	99.43
Con_6_2	1 206 840 250	24 136 805	1 200 300 350	24 006 007	99.45
Exp_45_1	1 206 808 350	24 136 167	1 200 167 600	24 003 352	99.44
Exp_45_2	1 206 837 050	24 136 741	1 200 482 550	24 009 651	99.47
Exp_6_1	1 206 850 000	24 137 000	1 200 301 150	24 006 023	99.45
Exp_6_2	1 018 654 150	20 373 083	1 013 573 600	20 271 472	99.50

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表 3 有效读段与参考基因组比对结果

Tab. 3 Clean reads mapped to reference genome

样品	总读段量	总映射读段占比/%	单一位点映射占比/%	多位点映射占比/%	未映射读段占比/%
Con_45_1	23 948 833	78.52	30.71	47.81	21.48
Con_45_2	23 943 524	80.59	30.67	49.92	19.42
Con_6_1	23 881 731	62.24	24.60	37.64	37.77
Con_6_2	24 006 007	76.02	31.18	44.84	23.99
Exp_45_1	24 003 352	42.84	16.69	26.15	57.16
Exp_45_2	24 009 651	53.74	20.94	32.80	46.26
Exp_6_1	24 006 023	75.69	30.36	45.33	24.31
Exp_6_2	20 271 472	75.19	30.54	44.65	24.81

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表 4 病毒感染6 h显著差异表达的氧化应激反应酶

Tab. 4 Oxidative stress enzymes that are significantly differentially expressed in 6 hpi

基因ID	log₂ (倍数)	P	描述
jgi\|Emihu1\|434150	−1.65	2.79×10⁻¹³	L-ascorbate peroxidase L-抗坏血酸过氧化物酶
jgi\|Emihu1\|444342	−2.36	3.57×10⁻¹⁹	L-ascorbate peroxidase L-抗坏血酸过氧化物酶
jgi\|Emihu1\|63449	9.58	1.24×10⁻²⁰	thioredoxin reductase 硫氧还蛋白还原酶
jgi\|Emihu1\|74843	−11.38	1.39×10⁻¹⁵	Thioredoxin 硫氧还蛋白
jgi\|Emihu1\|109275	−6.71	1.30×10⁻³	glutathione S-transferase 谷胱甘肽 S-转移酶
jgi\|Emihu1\|447453	−1.59	5.98×10⁻⁶	glutathione-S-transferase 谷胱甘肽 S-转移酶
jgi\|Emihu1\|439607	7.16	2.10×10⁻⁵	glutathione dehydrogenase 谷胱甘肽脱氢酶
jgi\|Emihu1\|115948	−1.50	1.65×10⁻⁵	glutathionedehydrogenase 谷胱甘肽脱氢酶
注：hpi为感染后小时数。

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表 5 病毒感染45 h显著差异表达的氧化应激反应酶

Tab. 5 Oxidative stress enzymes that are significantly differentially expressed in 45 hpi

基因ID	log2 (倍数)	P	描述
jgi\|Emihu1\|103927	8.21	6.41×10⁻⁴	glutathione peroxidase 谷胱甘肽过氧化物酶
jgi\|Emihu1\|433534	6.03	1.87×10⁻¹⁶	glutathione peroxidase 谷胱甘肽过氧化物酶
jgi\|Emihu1\|464198	1.24	4.00×10⁻³	peroxidase/catalase 过氧化物酶/过氧化氢酶
jgi\|Emihu1\|241133	−8.61	1.89×10⁻⁵	cytochrome c peroxidase 细胞色素c过氧化物酶
jgi\|Emihu1\|74843	11.08	4.51×10⁻¹²	thioredoxin 硫氧还蛋白
jgi\|Emihu1\|59424	10.06	7.34×10⁻⁶	thioredoxin 硫氧还蛋白
jgi\|Emihu1\|198128	−1.25	5.00×10⁻⁴	thioredoxin 硫氧还蛋白
jgi\|Emihu1\|66962	1.49	6.48×10⁻⁵	glutathione S-transferase 谷胱甘肽 S-转移酶
jgi\|Emihu1\|446089	1.31	2.33×10⁻⁷	glutathione S-transferase 谷胱甘肽 S-转移酶
jgi\|Emihu1\|109275	−8.32	1.89×10⁻⁵	glutathione S-transferase 谷胱甘肽 S-转移酶
jgi\|Emihu1\|63987	−1.22	1.18×10⁻⁹	glutathione reductase 谷胱甘肽还原酶
jgi\|Emihu1\|63016	−1.03	7.00×10⁻³	glutathione reductase 谷胱甘肽还原酶
注：hpi为感染后小时数。

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