‘呼红’牡丹高温胁迫响应生理与转录组分析

doi:10.16420/j.issn.0513-353x.2023-0941

园艺学报 ›› 2024, Vol. 51 ›› Issue (12): 2800-2816.doi: 10.16420/j.issn.0513-353x.2023-0941

• 遗传育种·种质资源·分子生物学 • 上一篇下一篇

‘呼红’牡丹高温胁迫响应生理与转录组分析

卜文轩¹, 姚奕平¹, 黄宇², 杨星宇¹, 罗小宁¹, 张旻桓¹^,²^,^*(), 雷维群², 王政³, 田珈宁¹, 陈露洁¹, 秦莉萍²

¹中南林业科技大学风景园林学院，长沙 410004
²南宁学院艺术与设计学院，南宁 530200
³河南农业大学风景园林与艺术学院，郑州 450046

收稿日期:2024-03-01 修回日期:2024-09-03 出版日期:2024-12-25 发布日期:2024-12-13
通讯作者:
^* E-mail：38358073@qq.com
基金资助:
湖南省自然科学基金项目(2022JJ31010); 湖南省自然科学基金青年基金项目(2023JJ41035); 湖南省研究生创新基金项目(2023CX02013); 湖南省研究生创新基金项目(CX20240698); 中南林业科技大学引进人才科研启动基金项目(2019YJ037); 中南林业科技大学研究生科研创新基金项目(2024CX02014); 国家自然科学基金青年基金项目(32401639); 湖南省林业科技攻关与创新项目(XLK202432); 湖南省教育厅重点项目(24A0195)

Transcriptome Analysis of the Response of Tree Peony‘Hu Hong’Under High Temperature Stress

BU Wenxuan¹, YAO Yiping¹, HUANG Yu², YANG Xingyu¹, LUO Xiaoning¹, ZHANG Minhuan¹^,²(), LEI Weiqun², WANG Zheng³, TIAN Jianing¹, CHEN Lujie¹, QIN Liping²

¹College of Landscape Architecture，Central South University of Forestry and Technology，Changsha 410004，China
²College of Art and Design，Nanning University，Nanning 530200，China
³College of Landscape Architecture and Art，Henan Agricultural University，Zhengzhou 450046，China

Received:2024-03-01 Revised:2024-09-03 Published:2024-12-25 Online:2024-12-13

摘要/Abstract

摘要：

为探究中国南方湿热地区本土牡丹（Paeonia suffruticosa）品种高温胁迫阶段的分子调控机制，以江南牡丹品种‘呼红’2 ~ 3年生植株为试材，采集20 ℃常温对照与40 ℃高温处理0、2、6、12和24 h的成熟叶片，采用生理指标和转录组分析方法探究高温胁迫下不同处理时间基因表达的差异。试验结果表明，随着高温胁迫时间的延长，叶片失水，生物膜受损，相对电导率和脯氨酸含量显著增加，明确了游离脯氨酸、相对电导率与可溶性蛋白为衡量牡丹高温胁迫响应中生理机制变化的重要指标。转录组测序总计产生492 693个unigene和840 509个转录本，36 174条差异表达基因在GO、KEGG等5个数据库得到注释。GO富集分析表明，高温处理对比组中热的反应途径、还原型戊糖磷酸循环途径等是主要的富集途径。KEGG富集分析表明，参与信号传导、光合作用、新陈代谢等相关的差异表达基因在响应高温胁迫中起主要作用。通过转录组分析，挖掘到多个参与耐热胁迫响应的转录因子和重要基因，包括热激转录因子PsHSFA2b、PsHSFA3、PsHSFB2b/2a、PsHSFB4、PsHSFC1和NAC、MYB、bHLH等家族基因。对选取的11个基因通过qRT-PCR检测在高温胁迫下的表达，其结果与RNA-seq结果相似。

关键词: 牡丹, 高温胁迫, 转录组, 生理生化指标

Abstract:

To analyze the molecular regulation mechanism of tree peony（Paeonia suffruticosa）in southern China，‘Hu Hong’was selected as the test material，the leaves treated at 20 ℃ and 40 ℃ were collected after 0，2，6，12 and 24 h，respectively. Transcriptomics and physiological and biochemical indicators were used to explore the differences in gene expression at different treatment times under high temperature stress. The result showed that the leaves showed water loss and biofilm damage，the relative conductivity of leaves and the proline content increased significantly with the prolongation of high temperature stress time. Free proline，relative conductivity and soluble protein were identified as important indicators to measure the changes of physiological mechanism in the response of peony to high temperature stress. A total of 492 693 unigene and 840 509 transcripts were generated by RNA-seq. 36 174 differentially expressed genes（DEGs）were annotated in 5 databases including GO and KEGG. The GO analysis showed that the main enrichment pathways were the reaction pathway to heat and the cycle pathway of reduced pentose phosphate. KEGG enrichment analysis showed that DEG is involved in signal transduction，photosynthesis and metabolism play a major role in responding to high temperature stress. Among the DEGs，multiple candidate genes and transcription factors involved in heat stress have been screened out，such as heat shock transcription factors PSHSFA2b，PSHSFA3，PSHSFB2a，PSHSFB2b，PSHSFB4，PSHSFC1 and some NAC，MYB，bHLH family genes. The expression of 11 selected genes under high temperature stress was detected by qRT-PCR，and the results were similar to RNA seq.

Key words: peony, high temperature stress, transcriptome, physiological and biochemical indicators

卜文轩, 姚奕平, 黄宇, 杨星宇, 罗小宁, 张旻桓, 雷维群, 王政, 田珈宁, 陈露洁, 秦莉萍. ‘呼红’牡丹高温胁迫响应生理与转录组分析[J]. 园艺学报, 2024, 51(12): 2800-2816.

BU Wenxuan, YAO Yiping, HUANG Yu, YANG Xingyu, LUO Xiaoning, ZHANG Minhuan, LEI Weiqun, WANG Zheng, TIAN Jianing, CHEN Lujie, QIN Liping. Transcriptome Analysis of the Response of Tree Peony‘Hu Hong’Under High Temperature Stress[J]. Acta Horticulturae Sinica, 2024, 51(12): 2800-2816.

图/表 14

表1 用于qRT-PCR的基因及其引物

Table 1 Genes and primers used for qRT-PCR

基因名称（编号） Gene name（number）	底物名称 Substrate name	引物序列（5′-3′） Primer sequence
HSP20（TRINITY_DN57811_c1_g1_i1）	S539-1	F：TGCCCAAAGAGGAAGAGAA；R：CCCATGCCATCAGAAATTAA
WRKY20（TRINITY_DN58753_c6_g1_i1）	S539-4-2	F：CTAATGGCATCACAACCGTC；R：ACTAGTTTTGACGCGTTTTGT
HSPA1s（TRINITY_DN58827_c1_g2_i1）	S539-3	F：TTGTCAGCCGGAGAAAGC；R：GCAGGAGGCGGAGAAGTA
HSFC1（TRINITY_DN58986_c1_g1_i2）	S539-4	F：AAGGCGGTGGTCTTGGTT；R：TGGGGGTGTTGAGTTGGA
HSFA3（TRINITY_DN61524_c1_g1_i2）	S539-5	F：CATTTTGGTGGTGCTTGACA；R：AGACGGTTTCGTTCTTGGC
CRF4（TRINITY_DN63834_c0_g1_i1）	S539-6	F：TTCAACAATCAACACTACGGG；R：AGAGTTAAGCACACGGGGTT
HSFB2b（TRINITY_DN64862_c0_g3_i2）	S539-7	F：GACTACTTTCGCAAGGGGA；R：TTACCGTCGTTGAAGGAGC
NAC55（TRINITY_DN66043_c1_g1_i3）	S539-8	F：ACAATACCAGTGCCGCTTC；R：CCCCTAATACCCCTAACGAC
MCU3（TRINITY_DN67541_c3_g1_i1）	S539-9	F：ATTAGGGAATTATTTTGGGGG；R：TGGAAGGTGATTTTTTGTGAA
HSP90B（TRINITY_DN71090_c1_g1_i1）	S539-10	F：AGGTCCTTGATTATGGGATGAT；R：TGGATGGTCTGCTAATATGGAA
HSP70（TRINITY_DN74057_c3_g1_i1）	S539-11-2	F：CTGGGACTTGCCGATGAC；R：ACGACACTGAGAAGGCCCT
内参 Reference	S539-DN59422	F：CGAATCTTGTCTTGACCCCC；R：ATTGTCACCACCATCCCTACC

图1 高温（40 ℃）处理下‘呼红’牡丹叶片表型、相对含水量与相对电导率的变化

Fig. 1 Changes of leaf phenotype，relative water content and relative conductivity of‘Hu Hong’peony under high temperature（40 ℃）treatment t-test，* α = 0.05.

图2 高温（40 ℃）处理下‘呼红’牡丹叶片叶绿素a、叶绿素b含量、叶绿素a/b和丙二醛含量的变化

Fig. 2 Changes of chlorophyll a，chlorophyll b content，chlorophyll a/b and malondialdehyde content in leaves of‘Hu Hong’peony under high temperature（40 ℃）treatment t-test，* α = 0.05.

图3 高温（40 ℃）处理下‘呼红’牡丹叶片可溶性糖、可溶性蛋白和游离脯氨酸含量变化

Fig. 3 Changes of soluble sugar，soluble protein and free proline content in leaves of‘Hu Hong’peony under high temperature（40 ℃）treatment t-test，* α = 0.05.

图4 高温（40 ℃）处理下‘呼红’牡丹叶片超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）活性的变化

Fig. 4 Changes of superoxide dismutase（SOD），peroxidase（POD）and catalase（CAT）activities in leaves of Paeonia suffruticosa‘Hu Hong’under high temperature（40 ℃）treatment t-test，* α = 0.05.

表2 ‘呼红’牡丹高温胁迫下生理指标相关性分析

Table 2 Correlation analysis of physiological indexes of‘Hu Hong’peony under high temperature stress

指标 Index	RWC	Rec	Chl	SS	SP	MDA	Pro	SOD	POD	CAT
RWC	1
Rec	-0.916^*	1
Chl	0.881^*	-0.852	1
SS	-0.924^*	0.922^*	-0.857	1
SP	-0.909^*	0.974^**	-0.764	0.947^*	1
MDA	-0.871	0.788	-0.943^*	0.917^*	0.763	1
Pro	-0.922^*	0.995^**	-0.812	0.911^*	0.983^**	0.751	1
SOD	0.748	-0.430	0.595	-0.635	-0.482	-0.731	-0.447	1
POD	-0.817	0.892^*	-0.557	0.771	0.929^*	0.504	0.931^*	-0.359	1
CAT	-0.690	0.914^*	-0.604	0.755	0.902^*	0.508	0.916^*	-0.066	0.897^*	1

表3 ‘呼红’牡丹高温胁迫下生理指标主成分分析结果

Table 3 Principal component analysis results of physiological indexes of‘Hu Hong’peony under high temperature stress

生理指标 Physiologic indexs	特征向量 Eigenvector
生理指标 Physiologic indexs	主成分1 Principal component 1	主成分2 Principal component 2
RWC	-0.968	0.180
Rec	0.981	0.173
Chl	-0.880	0.273
SS	0.968	-0.108
SP	0.975	0.174
MDA	0.865	-0.426
Pro	0.978	0.196
SOD	-0.594	0.719
POD	0.866	0.378
CAT	0.830	0.550
特征值 Characteristic value	8.058	1.361
方差贡献率/% Variance contribution rate	80.578	13.612
累积贡献率/% Cumulative contribution rate	80.578	94.191

图5 ‘呼红’牡丹高温（40 ℃）与常温（20 ℃）处理下差异表达基因火山图

Fig. 5 Volcano map of differentially expressed genes of‘Hu Hong’peony under high temperature（40 ℃）and normal temperature（20 ℃）treatment

图6 高温胁迫处理的‘呼红’牡丹叶片差异表达基因表达谱上方为趋势的ID，下方为趋势中的基因数量。

Fig. 6 Differentially expressed gene expression profiles of‘Hu Hong’peony leaves under high temperature stress Above is the ID of the trend，and below is the number of genes in the trend.

图7 ‘呼红’牡丹高温（40 ℃）处理与对照（20 ℃）同时间段差异表达基因GO富集分析气泡图

Fig. 7 GO enrichment analysis bubble diagram of differentially expressed genes between high temperature（40 ℃）treatment and control（20 ℃）at the same time period of‘Hu Hong’peony

图8 ‘呼红’牡丹高温（40 ℃）处理与对照（20 ℃）不同时间段差异表达基因GO富集分析气泡图

Fig. 8 GO enrichment analysis bubble diagram of differentially expressed genes in different time periods of high temperature （40 ℃）treatment and control（20 ℃）of‘Hu Hong’peony

图9 ‘呼红’牡丹高温（40 ℃）处理与对照（20 ℃）同时间段差异表达转录因子数量热图

Fig. 9 Heat map of differentially expressed transcription factors between high temperature（40 ℃）treatment and control（20 ℃）at the same time period of‘Hu Hong’peony

图10 ‘呼红’牡丹不同温度下差异表达转录因子基因表达图谱

Fig. 10 Differentially expressed transcription factor gene expression profiles of‘Hu Hong’peony at different temperatures

图11 ‘呼红’牡丹响应高温胁迫转录组数据的qRT-PCR验证

Fig. 11 qRT-PCR verification of transcriptome data of‘Hu Hong’peony in response to high temperature stress

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	赵溪, 张婷婷, 邢文婷, 王健, 宋希强, 周扬. 2021. 铁皮石斛热激蛋白HSP70基因家族鉴定及温度胁迫下的表达分析. 园艺学报, 48 (9):1743-1754. doi: 10.16420/j.issn.0513-353x.2020-0710
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‘呼红’牡丹高温胁迫响应生理与转录组分析

Transcriptome Analysis of the Response of Tree Peony‘Hu Hong’Under High Temperature Stress

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‘呼红’牡丹高温胁迫响应生理与转录组分析

Transcriptome Analysis of the Response of Tree Peony‘Hu Hong’Under High Temperature Stress

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参考文献 23

相关文章 15

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