泰州鹅开产前后肝脏组织转录组对比分析

张蕾; 章敬旗; 章玮月; 糜娟; 何文杰; 王健

doi:10.19303/j.issn.1008-0384.2020.09.010

泰州鹅开产前后肝脏组织转录组对比分析

doi: 10.19303/j.issn.1008-0384.2020.09.010

江苏农牧科技职业学院，江苏　泰州　225300

基金项目: 江苏省农业科技自主创新资金项目（CX[18]1004）；泰州市科技支撑计划（农业）项目（TN201802）

详细信息

作者简介:
张蕾（1987−），女，博士研究生，研究方向：动物遗传育种与繁殖（E-mail：leizhang@jsahvc.edu.cn）

通讯作者:
王健（1974−），男，教授，研究方向：动物遗传育种与繁殖（E-mail：568418583@qq.com）

中图分类号: S 835
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- 文章访问数: 1181
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- 被引次数: 0
出版历程
- 收稿日期: 2020-04-21
- 修回日期: 2020-07-23
- 刊出日期: 2020-09-28

RNA-Seq Analysis on Liver of Taizhou Geese before and after Egg-laying

Jiangsu Agri-animal Husbandry Vocational College, Taizhou, Jiangsu　225300, China

摘要

摘要: 目的进一步完善泰州鹅基因组结构信息，探索泰州鹅开产前、后肝脏的调控机制。方法利用RNA-Seq技术对开产前、后泰州鹅肝脏组织转录组进行分析，利用qRT-PCR对部分RNA-Seq数据进行验证；利用GO富集分析和KEGG通路注释对差异表达基因功能进行解析。结果测序获得的初始数据（raw reads）进行系列质控筛选，6个泰州鹅开产前、后肝脏组织获得73 596 894～79 837 756个高质量数据（clean reads），结合FPKM值定量估计所检测的基因表达值，筛选出202个差异基因，其中上调差异表达基因100个，下调差异表达基因102个，经GO功能注释后富集到42个生物功能类别；KEGG分析筛选到63条差异信号通路，其中5条显著富集信号通路为药物代谢-细胞色素P450、氮代谢、戊糖和葡萄糖醛酸相互转化、视黄醇代谢、类固醇激素生物合成通路，这些信号通路功能分析显示与脂质代谢以及生殖过程密切相关。结论开产前后泰州鹅肝脏组织RNA-Seq分析表明，泰州鹅开产前后肝脏组织中的差异基因多集中于脂质代谢以及生殖过程，研究结果为进一步研究泰州鹅开产期的肝脏调控机制以及加速泰州鹅地方种群的进一步选育提供了新的数据支撑。
- 转录组测序 /
- 泰州鹅 /
- 肝脏 /
- 开产
Abstract: Objective Regulating functions of goose liver were studied by genetic analyses before and after the birds started egg-laying. Method RNA-Seq technology was employed to obtain the transcriptome of the liver tissue of Taizhou goose before and after egg-laying. The results were verified using a qRT-PCR method and followed by the Go annotation and KEGG analysis to determine the functions of the differentially expressed genes (DEGs). Result After an initial quality control screening on the raw RNA-Seq data, 73 596 894 to 79 837 756 clean reads were secured from 6 liver specimens. Based on the estimated quantitative FPKM value, 100 upregulated and 102 downregulated genes were identified. By GO annotation, all DEGs were enriched into 42 categories of biological functions. The KEGG analysis found 63 pathways including 5 significant ones that associated with the drug metabolism-cytochrome P450, nitrogen metabolism, pentose and glucuronate interconversions, retinol metabolism, and steroid hormone biosynthesis. These pathways are known to closely relate to lipid metabolism and reproduction regulation. Conclusion The RNA-Seq transcriptomes on the Taizhou goose livers sampled before and after the start of egg-laying indicated that the enriched DEGs mostly involved the lipid metabolism and reproduction process. The information would support further studies on and breeding of Taizhou geese.
- RNA-Seq /
- Taizhou goose /
- liver /
- egg-laying

HTML全文

图 1 转录组数据比对基因区域分布统计

注：1～6：样品编号；不同颜色代表所在基因的不同区域（外显子、内含子以及基因间区），区域大小代表具有某一覆盖度范围的转录组数据所占比例。

Figure 1. Transcriptome data vs. regional distribution of genes

Note: 1~6: sample codes; different colors in pie chart represent different locations of genes (exon, intron and intergenic); area size represents transcriptome datum coverage.

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图 2 差异表达基因火山图

注：差异表达火山图中的每一个点表示一个基因。蓝色代表下调表达基因，黄色代表上调表达基因，灰色代表无显著性差异表达的基因。

Figure 2. Volcano plot of differentially expressed genes

Note: each dot represents a gene. Blue dot: significantly downregulated genes; yellow dot: significantly upregulated genes; gray dot: genes with no differentiated expression.

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图 3 差异表达基因热图聚类分析

注：图中1～6列代表不同的样品，不同行代表不同的基因。颜色代表了基因在样品中的表达量FPKM的对数值，蓝色代表下调表达基因，红色代表上调表达基因。

Figure 3. Heat map of differentially expressed genes

Note: Columns 1–6: individual samples; different rows represent differentially expressed genes. Color scales as log₂ (FPKM); blue: significantly downregulated genes; red: significantly upregulated genes.

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图 4 开产前后泰州鹅肝脏组织相关基因表达检测

Figure 4. Expressions of related genes in liver of Taizhou geese before and after start of egg-laying

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图 5 差异表达基因GO注释

注：细胞组分：胞外区、细胞、膜、细胞连接处、膜封闭腔、大分子复合物、细胞器、胞外区部分、细胞器部分、病毒离子部分、膜部分、突触部分、细胞部分、突触，胞上、分子复合物；生物过程：免疫系统过程、行为、代谢过程、细胞增殖、细胞过程、生殖过程、生物黏附、信号传导、多细胞组织过程、发育过程、生长、运动、节律过程、刺激反应、定位、多生物过程、生物调节、细胞成分组织或生物发生；分子功能：催化活性、信号转导活性、结构分子活性、转运体活性、结合、翻译调节活性、分子转导活性、分子功能调节、转录、调节活性。

Figure 5. GO annotation on differentially expressed genes

Note：Cellular component: extracellular region, cell, membrane, cell junction, membrane-enclosed lumen, macromolecular complex, organelle, extracellular region part, organelle part, virion part, membrane part, synapse part, cell part, synapse, supramo, lecular complex；Biological process：immune system process, behavior, metabolic process, cell proliferation, cellular process, reproductive process, biological adhesion, signaling, multicellular organismal process, developmental process, growth, locomotion, rhythmic process, response to stimulus, localization, multi-organism process, biological regulation, cellular component organization or biogenesis；Molecular function：catalytic activity, signal transducer activity, structural molecule activity, transporter activity, binding, translation regulator activity, molecular transducer activity, molecular function regulator, transcription, regulator activity.

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图 6 KEGG富集分析

Figure 6. KEGG enrichment on differentially expressed genes

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表 1 qRT-PCR引物序列、退火温度以及目的片段大小

Table 1. Primer sequences, annealing temperature, and predicted length for qRT-PCR

基因 Gene	引物序列（5′-3′） Sequence （5′-3′）	退火温度 Tm/℃	产物大小 Size/bp
CHRNA9	F:CAGCCAAAGGGAAATATGCTC	60	180
CHRNA9	R:TTTATCCGTGTCTTCCACTGGTC	60	180
MRAP	F:ATTCACTCTTCTCTCAGTTACGTC	58	135
MRAP	R:AACATGAGGAGTAACTGGTACATG	58	135
EPSTI1	F:GGAGGAAATGAAGCAAGAACAAC	60	160
EPSTI1	R:TCCTCTGTTCATTTTGATGTGCTC	60	160
TLDC2	F:AGCCCTGGAGCCTGCTGTAC	60	152
TLDC2	R:GCCTCAGTGTCACGGATGAG	60	152
SLC13A5	F:GTCTTCACCGAATGCACCAG	61	128
SLC13A5	R:AGCATGAAGGCAAAGGAAGCAC	61	128
β-actin	F:CAGCCATCTTTCTTGGGTAT	60	164
β-actin	R:CTGTGATCTCCTTCTGCATCC	60	164

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表 2 样本数据质量预处理评价分析

Table 2. Quality evaluation and analysis on data preprocessing

样品编号 No.	Raw读数 Raw reads	Raw基数 Raw bases	净读数 Clean reads	净读数比例 Clean ratio/%	碱基质量 Q30/%
1	81 525 658	12 228 848 700	79 443 928	97.45	95.46
2	76 140 902	11 421 135 300	74 434 560	97.76	95.43
3	81 984 186	12 297 627 900	79 837 756	97.38	95.62
4	77 907 180	11 686 077 000	76 091 514	97.67	95.64
5	75 754 528	11 363 179 200	73 596 894	97.15	95.82
6	77 403 998	11 610 599 700	75 702 218	97.80	95.41
注：Raw读数：每个样本的测序raw reads的总数；Raw基数：每个样本测序所获碱基的总数；净读数：过滤后的测序数据；净读数比例：过滤后高质量序列数占原始下机序列数的比例；碱基质量：过滤后，总序列中质量值大于30（错误率小于0.1%）的碱基数的比例。 Note：raw reads: the number of raw reads; raw bases: the number of bases counted on raw reads; clean reads: the number of clean reads after preprocessing; clean ratio: the ratio of bases counted on clean reads; Q30: the ratio of clean bases whose quality threshold over 30.

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表 3 转录组数据与参考基因组比对结果统计

Table 3. Results of clean reads on sample vs. reference genome

样品编号 No.	总读数 Total reads	总覆盖数 Total mapped	覆盖率 Mapped ratio/%	多重覆盖数 MultiMap reads	多重覆盖率 MultiMap ratio/%
1	79 443 928	70 396 347	88.61	2 257 950	2.84
2	74 434 560	67 324 680	90.45	2 308 666	3.10
3	76 091 514	67 957 322	89.31	1 993 788	2.62
4	73 596 894	66 002 063	89.68	1 916 837	2.60
5	75 702 218	67 433 936	89.08	1 967 303	2.60
6	79 837 756	70 832 388	88.72	2 001 710	2.51
注：总读数：测序序列经过测序数据过滤后的数量（Clean reads）；总覆盖数：能定位到基因组上的测序序列的数量；覆盖率：在参考序列上能够比对位置的测序序列的百分比；多重覆盖数：比对到基因组多个位置的序列数；多重覆盖率：比对到基因组多个位置的序列数的百分比。 Note：total reads: the number of clean reads; total mapped: the number of clean reads that align with reference genome; mapped ratio: the percentage of clean reads that align with reference genome; multiMap reads: the number of clean reads that align with reference genome at multiple positions; multiMap ratio: the percentage of clean reads that align with reference genome at multiple positions.

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表 4 泰州鹅开产前、后肝脏组织上调/下调差异最大的前10位基因

Table 4. Top 10 upregulated and downregulated genes in Taizhou geese before and after start of egg-laying

基因名称 Gene ID	开产前表达量 Before laying	开产后表达量 Laying group	相对表达量 Log₂FC	上调/下调 Up/Down	基因描述 Gene description
gene10578	150.00	83.89	−2.52	下调 Down	CHRNA9
gene14778	10698.00	6660.66	−2.07	下调 Down	SLC13A5
gene20392	1892.67	1136.15	−2.06	下调 Down	RGS9BP
gene14476	1484.33	893.87	−1.94	下调 Down	MRAP
gene6885	94.00	509.69	2.51	上调 Up	EPSTI1
gene3244	10.33	58.45	2.51	上调 Up	MYO18B
gene4794	292.00	1268.87	2.37	上调 Up	LOC106032697
gene16287	20.67	85.35	2.15	上调 Up	TLDC2
gene9668	89.33	397.50	2.09	上调 Up	LOC106038139
gene12640	22.67	130.54	1.99	上调 Up	TMEM132E

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表 5 泰州鹅开产前、后肝脏组织富集显著性最显著的前5条通路

Table 5. Top 5 enriched pathways in liver of Taizhou geese before and after start of egg-laying

通路名称 Pathway name	通路编号 Pathway ID	富集基因数目 Count	校正后P值 P value
Drug metabolism - cytochrome P450 药物代谢-细胞色素P450信号通路	map00982	3	0.01
Nitrogen metabolism 氮代谢信号通路	map00910	5	0.02
Pentose and glucuronate interconversions 戊糖和葡萄糖醛酸相互转化信号通路	map00040	8	0.03
Retinol metabolism 视黄醇代谢信号通路	map00830	3	0.01
Steroid hormone biosynthesis 类固醇激素生物合成信号通路	map00140	6	0.01

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