The Mechanism of Heat Stress Response and the Exploration of Functional Genes in Rosa chinensis‘Angela’

doi:10.16420/j.issn.0513-353x.2023-0417

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (6): 1284-1296.doi: 10.16420/j.issn.0513-353x.2023-0417

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

The Mechanism of Heat Stress Response and the Exploration of Functional Genes in Rosa chinensis‘Angela’

YANG Binan¹, LI Bowen¹, YANG Zhenyu¹, XU Yipeng¹, YAN Yunqing¹, LOU Yuxia¹, FENG Shucheng²^,^**(), MING Feng¹^,^**()

¹ Plant Germplasm Resource Development Collaborative Innovation Center，College of Life Sciences，Shanghai Normal University，Shanghai 200234，China
² Shanghai Greening Management Guidance Station，Shanghai 200020，China

Received:2024-03-13 Revised:2024-05-27 Online:2024-12-18 Published:2024-06-22
Contact: FENG Shucheng, MING Feng

Abstract

Abstract:

In this study，high temperature experiment was conducted and phylogenetic index were analyzed using Rosa chinensis‘Angela’‘Renyue’and‘Xianjing’as test materials. The results showed that‘Angela’exhibited the highest heat resistance among the three varieties. Comparative transcriptome analysis，co-expression network analysis，protein-protein interaction network analysis and phylogenetic analysis revealed that the improvement of heat tolerance in‘Angela’is associated with the overexpression of heat shock transcription factors（HSFs）and heat shock proteins（HSPs）. Notably，the expression level of HSP gene RcHSP18.1 significantly increased with prolonged heat stress duration. Furthermore，by silencing the homologous gene NtHSP18.1 in tobacco，the heat resistance of tobacco was significantly reduced，confirming the essential role of HSP18.1 in heat stress tolerance. Overexpressing of RcHSP18.1 through gene bombardment and silencing RcHSP18.1 gene by virus induced gene silencing technique have both confirmed the significant role the gene RcHSP18.1 in enhancing heat stress resistance in‘Angela’.

Key words: rose, heat stress, gene, function

YANG Binan, LI Bowen, YANG Zhenyu, XU Yipeng, YAN Yunqing, LOU Yuxia, FENG Shucheng, MING Feng. The Mechanism of Heat Stress Response and the Exploration of Functional Genes in Rosa chinensis‘Angela’[J]. Acta Horticulturae Sinica, 2024, 51(6): 1284-1296.

Figures/Tables 10

Table 1 Primers information in this study

用途 Application	名称 Name	引物序列（5′-3′） Primer sequence
基因克隆 Gene clone	RcHSP18.1	F：ATGTCGCTCATCCCAAATTTCC R：TTACCCGGAAATTTCAATGGC
酵母双杂交 Yeast two hybrid	AD-HSP18.1	F：ATGGCCATGGAGGCCAGTGAATTCATGTCGCTCATCCCAAATTTCC R：CTGCAGCTCGAGCTCGATGGATCCTTACCCGGAAATTTCAATGGC
	BD-HSP18.1	F：CATATGGCCATGGAGGCCGAATTCATGTCGCTCATCCCAAATTTCC R：CGGCCGCTGCAGGTCGACGGATCCTTACCCGGAAATTTCAATGGC
	AD-HSF30	F：ATGGCCATGGAGGCCAGTGAATTCATGGAGGGAGTAGCAGTGAAAG R：CTGCAGCTCGAGCTCGATGGATCCTCAAGGATTGGGCTTGATCAGAAA
	BD-HSF30	F：CATATGGCCATGGAGGCCGAATTCATGGAGGGAGTAGCAGTGAAAG R：CGGCCGCTGCAGGTCGACGGATCCTCAAGGATTGGGCTTGATCAGAAA
亚细胞定位和过表达验证 Subcellular localization and overexpression verification	pCAMBia-2300-GFP- RcHSP18.1	F：GGATCCTCTAGAACTAGTCATGTCGCTCATCCCAAATTTCC R：GTCAGAAGGCCTCCCGGGTTACCCGGAAATTTCAATGGC
基因沉默 Gene silencing	TRV2-HSP18.1-1	F：TGAGTAAGGTTACCGAATTCGCATCTTCGACCTCGATCTCTGG R：GACATGCCCGGGCCTCGAGTGGAGAACTTGCCGCTGCTT
	TRV2-HSP18.1-2	F：TGAGTAAGGTTACCGAATTCCGGATCGACTGGAAGGAGACC R：GACATGCCCGGGCCTCGAGGACATCAGGCCTCTTCACCTCTC
qRT-PCR	qPCR-HSP18.1	F：GTCGCTCATCCCAAATTTCC R：TTCTCGCCAGGAAATTCAGG
	qPCR-HSF30	F：CATCAAGAGGAGGAGGCATGTGTCG R：GTCTTTCAAGCTCAGTCTCCAGCCC

Fig. 1 ‘Angela’‘Renyue’and‘Xianjing’rose cultivars phenotypes following 2-hour treatments at different temperatures（A），DAB and NBT staining（B），and relative electrical conductivity（C） DAB：Diaminobenzidine staining，with darker color indicating higher SOD activity；NBT：Nitro blue tetrazolium staining，where deeper color reflects higher levels of reactive oxygen species. * indicated significant differences at 0.05 level.

Fig. 2 GO analysis and KEGG pathway enrichment analysis of differentially expressed genes in‘Angela’after 2 hours of treatment under high temperature（46 ℃）and normal temperature（25 ℃）conditions

Table 2 Differentially expressed heat stress-related genes in‘Angela’after 2-hour treatments at high temperature（46 ℃）compared to normal temperature（25 ℃）

基因类别 Gene category	基因识别数量 Identified	差异表达基因数量 Differenced	表达量上升数量 Up regulated	表达量下降数量 Down regulated
热信号转导 Heat signal transduction	32	5	0	5
转录调控 Transcription regulation	42	22	18	4
蛋白质稳态 Protein homeostasis	50	17	16	1
活性氧稳态 ROS homeostasis	27	3	1	2
RNA稳态 RNA homeostasis	1	1	1	0
基因总数 Total quantities of gene	152	48	36	12

Fig. 3 In‘Angela’rose，analysis of RcHSP18.1 expression levels under normal and high temperature conditions，along with the candidate regulatory gene network analysis A：Heatmap analysis of differentially expressed heat-responsive genes（HRGs）；B：Expression analysis of heat shock transcription factors（HSFs）before and after heat stress；C：Interaction network between heat shock transcription factor HSF and heat shock protein HSP genes.

Fig. 4 In‘Angela’，under high temperature conditions，the phenotypic traits as well as the expression levels and subcellular localization of the RcHSP18.1 gene A：Phenotypic changes；B：Changes in RcHSP18.1 expression levels detected by qRT-PCR；C：Transcriptomic data showing expression levels of RcHSP18.1，*** indicated significant differences at 0.001 level；D：Subcellular localization of RcHSP18.1 in epidermal cells of the leaf.

Fig. 5 Phenotype of tobacco after silencing HSP18.1（A），changes in relative electrical conductivity and gene expression levels（B），and DAB and NBT staining（C） HSP18.1：Gene silenced strain；GFP：Empty vector control；DAB：Diaminobenzidine staining，where darker color indicates higher SOD activity；NBT：Nitro Blue Tetrazolium staining，with deeper color reflecting higher levels of reactive oxygen species. * indicated significant differences at 0.05 level.

Fig. 6 The comparison of RcHSP18.1 expression levels and relative electrical conductivity between overexpression lines（OX）and wild-type（WT）of RcHSP18.1 in callus tissue of‘Angela’rose * indicated significant differences at 0.05 level；** indicated significant differences at 0.01 level.

Fig. 7 The phenotypic effects post-silencing of the RcHSP18.1 gene（A），changes in gene expression levels（B），and DAB and NBT staining results（C）in‘Angela’rose TRV2-HSP18.1：Gene silenced strain targeting HSP18.1；TRV2-GFP：Empty vector control expressing GFP；DAB：Diaminobenzidine staining，where darker color indicates higher SOD activity；NBT：Nitro blue tetrazolium staining，with deeper color reflecting higher levels of reactive oxygen species. *** indicated significant differences at 0.001 level.

Fig. 8 Identification of the expression pattern of RcHSF30 in response to heat stress and its interaction with HSP18.1 in‘Angela’rose ** indicated significant differences at 0.01 level.

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The Mechanism of Heat Stress Response and the Exploration of Functional Genes in Rosa chinensis‘Angela’

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