酸雨胁迫下琯溪蜜柚叶片转录组差异表达分析

张琼; 陆銮眉; 朱丽霞

doi:10.19303/j.issn.1008-0384.2021.07.003

酸雨胁迫下琯溪蜜柚叶片转录组差异表达分析

doi: 10.19303/j.issn.1008-0384.2021.07.003

张琼^{1, 2,},
陆銮眉^{1, 2},
朱丽霞^{1, 2}

1.
闽南师范大学生物科学与技术学院，福建　漳州　363000
2.
闽台特色园林植物福建省高等学校重点实验室，福建　漳州　363000

基金项目: 福建省自然科学基金项目（2020J01810）

详细信息

作者简介:
张琼（1980−），女，博士研究生，副教授，研究方向：植物生态学（E-mail：349369029@qq.com）

中图分类号: S 666.3；Q 948.1
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出版历程
- 收稿日期: 2021-03-07
- 修回日期: 2021-04-15
- 网络出版日期: 2021-07-13
- 刊出日期: 2021-07-28

Transcriptome Changes of Citrus grandis Seedlings in Response to Acid Rain Stress

ZHANG Qiong^{1, 2
,},
LU Luanmei^{1, 2},
ZHU Lixia^{1, 2}

1.
School of Biological Sciences and Biotechnology, Zhangzhou, Fujian　363000, China
2.
Minnan Normal University, Zhangzhou, Fujian　363000, China

摘要

摘要: 目的研究琯溪蜜柚受酸雨胁迫的内在分子机制，为琯溪蜜柚科学种植提供基础资料，也为酸雨逆境生理提供理论基础。方法以模拟酸雨胁迫24 h的琯溪蜜柚叶片进行Illumina HiSeq TM 4000 高通量转录组测序分析，将组装得到的基因在参考基因组、Nr和 KEGG 数据库进行比对；利用 FDR 与 log₂（FC）来筛选差异基因，筛选条件为 FDR＜0.05且|log₂（FC）|＞1，将筛选的差异基因做GO和KEGG富集分析。结果模拟酸雨喷淋24 h后，琯溪蜜柚嫩叶出现明显块状伤斑；共得到21497个基因，全部得到注释，与对照相比，酸雨处理组中有879个基因显著上调，588个基因显著下调；筛选出样本中前50个DEGs（差异基因），均为上调表达基因，大部分涉及代谢途径、次生代谢、苯丙基类丙烷生物合成和萜类物质合成等相关基因。GO富集分析表明差异表达基因主要位于细胞外区域；执行分子功能中催化剂活性是最为显著富集的GO term，其次是氧化还原酶活性；生物过程中差异表达基因最显著的GO term是DNA 代谢过程。KEGG富集分析表明DNA复制是差异表达基因中最显著富集的Pathway，其次是次生代谢的生物合成，再次是苯丙素类合成途径。对次生代谢合成途径中4个差异表达基因[POD同工酶cg3g018770和cg2g001440、肉桂酰辅酶A还原酶（CCR）同工酶cg1g021310、4-香豆酸-辅酶a连接酶（4CL）同工酶cg3g029290]进行PCR荧光定量分析，验证了转录组数据的可靠性。结论琯溪蜜柚对酸雨耐性较强，对酸雨胁迫的响应是多基因参与、多生物过程协同调控的过程，次生代谢的调节可能是应对酸雨胁迫的主要方式。
- 琯溪蜜柚 /
- 酸雨胁迫 /
- 转录组测序 /
- 差异表达基因
Abstract: Objective Molecular mechanisms of Citrus grandis (L.) Osbeck. cv. Guanximiyou in response to simulated acid rain stress were investigated. Method The llumina HiSeq 4000 system, a high-throughput transcriptome sequencing technology, was applied to reveal the differential expressions of the grapefruit transcriptome after a 24 h simulated acid rain treatment. The unigenes obtained were compared to the Nr and KEGG database. Abundance of gene expression of the samples were screened according to transcriptome data by using PRKM method. Differentially expressed genes (DEGs) among the treated samples were estimated by referring to the standard of FDR ≤ 0.05 and |log₂FC| ≥ 1. Functions and pathways of those DEGs were analyzed using the Gene Ontology (GO) and KEGG pathway database. Result Significant lumpy lesions began to appear on the young grapefruit leaves 24 h after the artificial acid rain spray with 21 497 fully described unigenes obtained. In comparison to control, 879 of the genes were significantly upregulated and 588 downregulated. The top 50 DEGs were all in the upregulated category and mainly associated with metabolic pathways, secondary metabolism, phenylpropyl propane biosynthesis, or terpenoid biosynthesis. The GO enrichment analysis showed the DEGs being largely located in extracellular region and, among various molecular functions, the catalytic activity being the most significantly enriched, followed by oxidoreductase activity, while the DNA metabolism being the most significant of DEGs in the GO term on biological process. The KEGG enrichment analysis indicated that DNA replication was the most significant enrichment pathway, followed by secondary metabolic biosynthesis, and lignin synthesis. The PCR fluorescence quantitative analysis on the 4 DEGs in the secondary metabolic biosynthesis pathway [i.e., POD isozyme cg3g018770 and cg2g001440, cinnamyl coenzyme A reductase (CCR) isozyme cg1g021310, and 4-coumaric acid-coenzyme A ligase (4CL) isozyme cg3g029290] confirmed that the acid rain stress indeed significantly affected the expressions of these genes. Conclusion C. grandis (L.) Osbeck. cv. Guanximiyou was strongly tolerant to acid rain. The stress response of the plants involved numerous genes regulated by various collaborative biological processes. Among them, the regulation of secondary metabolism appeared to play a major role in coping with acid rain stress by the plant.
- Citrus grandis (L.) Osbeck. cv. Guanximiyou /
- acid rain stress /
- transcriptome sequencing /
- differentially expressed genes

HTML全文

图 1 模拟酸雨喷淋对叶片伤害表型

Figure 1. Phenotype effect of water or acid rain spraying on leaves

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图 2 样本表达量的层级聚类

Figure 2. Hierarchical cluster diagram on expression of sample

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图 3 KEEP 代谢途径中最为显著富集的前20条通路

注：Rich factor指差异表达的基因中位于该pathway条目的基因数目与所有基因中位于该pathway条目的基因总数的比值， Rich factor越大，表示富集的程度越高。Q value是做过多重假设检验校正之后的P value，取值范围为0到1，越接近于零，表示富集越显著。

Figure 3. Top 20 enrichment pathways as analyzed by KEEP

Note: Rich factor refers to ratio of number of DEGs located in pathway entry to number of all genes located in same pathway entry; high rich factor indicates high degree of enrichment. Q value is the P value after multiple hypothesis testing and corrections which ranges from 0 to 1 with 0 being most significantly enriched.

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图 4 POD同工酶,CCR同工酶和4CL同工酶基因相对表达量

Figure 4. Relative expressions of POD, CCR, and 4CL isoenzyme genes

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表 1 qPCR 反应体系

Table 1. qPCR reaction system

qPCR反应体系 qPCR reaction system	体积 Volume/μl
qPCR反应混合物 qPCR Master Mix	10
PCR正向引物PCR Forward Primer/（10 mol·L⁻¹）	0.4
PCR反向引物 PCR Reverse Primer/（10 mol·L⁻¹）	0.4
cDNA模板 cDNA template	4
ddH₂O	5.2
合计 Total	20

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表 2 引物序列

Table 2. Primer sequence

基因 Gene		序列 Sequence
Cg3g024100	F	5′CAATGTGAAGTCCAGCGTGTG3′
Cg3g024100	R	5′AGCCCTCGTAGTTCTCGTCA3′
Cg3g018770	F	5′CATTTACCAATCGCCTCTATCC3′
Cg3g018770	R	5′ACCCCTGTCGGTTCATCAAGT3′
Cg2g001440	F	5′GGCAATCCAGACCCAACACT3′
Cg2g001440	R	5′AATGGCAGCGGTATCGGC3′
Cg1g021310	F	5′GTTGACGAACATTGCTGGAGT3′
Cg1g021310	R	5′GCAGCAATGTCCCTATCACC3′
Cg3g029290	F	5′CGAAGTAGAGTCCCTCAAGCA3′
Cg3g029290	R	5′TGTCACCAGTATGAAGCCAACC3′

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表 3 有效数据评估统计

Table 3. Statistics of valid data

样本 Sample	处理后序列数据 Clean Data/bp	过滤后的序列数据 HQ Clean Data/bp	处理后序列数 Clean Reads Num/条	过滤后的序列 HQ Clean Reads		Q20		Q30		GC
样本 Sample	处理后序列数据 Clean Data/bp	过滤后的序列数据 HQ Clean Data/bp	处理后序列数 Clean Reads Num/条	数量 Number/条	占比 Ratio/%	数量 Number/bp	占比 Ratio/%	数量 Number/bp	占比 Ratio/%	数量 Number/bp	占比 Ratio/ %
CK-1	7361352600	7111541211	49075684	48127600	98.07	6999438082	98.42	6769749153	95.19	3153041858	44.34
CK-2	8465603100	8202153477	56437354	55469140	98.28	8080142163	98.51	7826038154	95.41	3620789302	44.14
CK-3	8683275600	8408343801	57888504	56867832	98.24	8281909562	98.5	8019114646	95.37	3726434942	44.32
T-1	8203212900	7937070962	54688086	53695546	98.19	7816272312	98.48	7566409577	95.33	3522622180	44.38
T-2	9249897000	8926570988	61665980	60549386	98.19	8792579583	98.5	8514792020	95.39	3962645890	44.39
T-3	7880490300	7621577079	52536602	51567848	98.16	7503238757	98.45	7259492038	95.25	3369836120	44.21
注：1.Q20-碱基质量 ≥20的碱基；2.Q30-碱基质量 ≥30的碱基；3.GC-鸟嘌呤和胞嘧啶；4.CK-1、CK-2、CK-3为清水对照3个重复，T-1、T-2、T-3为pH2.5模拟酸雨喷淋的3个重复，表4、5同，图2、4同。 Note: 1. Q20- Base quality≥20. 2. Q30- Base quality≥30.3. GC- Guanine and Cytosine.4.CK-1, ck-2 and ck-3 are three repetitions of clear water control, and T-1, T-2 and T-3 are three repetitions of pH2.5 simulated acid rain spray.The same as Tab. 4 and. The same as Fig.2 and 4.

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表 4 过滤后的序列与核糖体RNA 的比对统计

Table 4. Statistics of HQ-clean data and rRNA

样品 Sample	过滤后的序列数 HQ Clean Reads Number/条	比对上rRNA的序列 Mapped Reads		未比对上rRNA的序列 Unmapped Reads
样品 Sample	过滤后的序列数 HQ Clean Reads Number/条	数量 Number/条	占比 Ratio/%	数量 Number/条	占比 Ratio/%
CK-1	48127600	370958	0.77	47756642	99.23
CK-2	55469140	435276	0.78	55033864	99.22
CK-3	56867832	597704	1.05	56270128	98.95
T-1	53695546	404974	0.75	53290572	99.25
T-2	60549386	595394	0.98	59953992	99.02
T-3	51567848	327954	0.64	51239894	99.36

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表 5 比对核糖体后得到的未标记序列与参考基因组的比对统计

Table 5. Statistics of unmapped reads and reference genome

样品 Sample	序列总数 Total reads/条	唯一比对上的序列 Unique mapped reads		多处比对上的序列 Multiple mapped reads
样品 Sample	序列总数 Total reads/条	数量 Number/条	占比 Ratio/%	数量 Number/条	占比 Ratio/%
CK-1	47756642	41591814	87.09	672956	1.41
CK-2	55033864	47903535	87.04	849190	1.54
CK-3	56270128	48813881	86.75	868458	1.54
T-1	53290572	46327128	86.93	743356	1.39
T-2	59953992	51103454	85.24	834008	1.39
T-3	51239894	44511502	86.87	705832	1.38

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表 6 差异基因GO功能分类

Table 6. The Go functional classification of the differential expression

本体 Ontology	分类 Class	上调表达基因数量 Up-regulated DEG number	下调表达基因数量 Down-regulated DEG number	本体 Ontology	分类 Class	上调表达基因数量 Up-regulated DEG number	下调表达基因数量 Down-regulated DEG number
生物过程 Biological Process	单一的生物过程 single-organism process	90	45	分子功能 Molecular Function	催化剂活性 catalytic activity	163	81
	细胞杀伤 cell killing	1	0		转运因子活性 transporter activity	19	3
	刺激应答 response to stimulus	24	7		信号转导过程 signal transducer activity	5	2
	细胞成分组织 cellular component organization or biogenesis	8	12		分子功能调节 molecular function regulator	1	1
	免疫系统过程 immune system process	2	0		核苷酸结合转录因子活性 nucleic acid binding transcription factor activity	3	0
	多组织过程/多机体过程 multi-organism process	5	0		结合/结合剂活性 binding	74	54
	定位 localization	29	5		结构分子活性 structural molecule activity	1	0
	再生 reproduction	4	1	细胞成分 Cellular Component	细胞间区域 extracellular region	6	2
	生长 growth	0	1		细胞连丝 cell junction	4	0
	生物调控 biological regulation	13	7		细胞膜要素 membrane part	36	5
	再生过程 reproductive process	2	0		细胞膜 membrane	38	6
	多细胞组织过程 multicellular organismal process	1	2		膜结合腔体 membrane-enclosed lumen	0	2
	发育过程 developmental process	2	2		细胞器 organelle	11	23
	细胞过程 cellular process	78	64		细胞 cell	23	26
	信号 signaling	2	2		细胞要素 cell part	23	26
	代谢过程 metabolic process	116	68		大分子复合物 macromolecular complex	3	8
					细胞器要素 organelle part	2	4

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