月季‘安吉拉’热胁迫反应机制及功能基因的挖掘

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

摘要/Abstract

摘要：

以月季品种‘安吉拉’‘韧月’‘仙境’为材料，模拟高温处理（46 ℃，4 h），通过表型以及生理指标分析，明确‘安吉拉’对热胁迫的抵御能力最强。通过转录组、共表达、蛋白互作网络以及多态性分析发现，‘安吉拉’较高的热胁迫抵御能力与热激蛋白（heat shock protein，HSP）以及热激转录因子（HSF）家族成员基因的超量表达有关。其中月季热激蛋白基因RcHSP18.1 的表达水平受高温诱导提高最为显著。在烟草中瞬时沉默HSP18.1后发现，HSP18.1蛋白的表达能够显著提高宿主热胁迫的抵御能力。采用基因枪法对‘安吉拉’月季愈伤组织进行RcHSP18.1过表达以及对植株进行病毒诱导基因沉默，均验证RcHSP18.1对抵御热胁迫提高发挥了重要作用。

关键词: 月季, 热胁迫, 基因, 功能

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

杨碧楠, 李博文, 杨振宇, 徐奕芃, 阎韵清, 娄玉霞, 奉树成, 明凤. 月季‘安吉拉’热胁迫反应机制及功能基因的挖掘[J]. 园艺学报, 2024, 51(6): 1284-1296.

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.

图/表 10

表1 本研究中所用引物信息

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

图1 ‘安吉拉’‘韧月’和‘仙境’月季不同温度处理2 h后的表型（A）、DAB与NBT染色（B）和相对电导率（C） DAB：二氨基联苯胺染色，颜色深表示SOD活性高；NBT：硝基蓝四氮唑染色，颜色深显示活性氧含量高。* 表示在0.05水平差异显著。

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.

图2 ‘安吉拉’月季高温（46 ℃）与常温（25 ℃）处理2 h后的差异表达基因的GO分析和KEGG通路富集分析

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

表2 ‘安吉拉’月季高温（46 ℃）与常温（25 ℃）处理2 h后差异表达的热应激相关基因

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

图3 ‘安吉拉’月季正常和高温下RcHSP18.1表达水平和候选调控基因网络分析 A：差异表达热相关基因（HRG）的热图分析；B：热胁迫前后热激转录因子（HSF）的表达分析；C：热激转录因子HSF和热激蛋白HSP基因间的相互作用网络。

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.

图4 ‘安吉拉’月季在高温下的表型和RcHSP18.1表达水平及亚细胞定位 A：表型变化；B：qRT-PCR检测的RcHSP18.1表达量变化；C：转录组数据中RcHSP18.1的表达量，*** 表示在0.001水平差异显著；D：RcHSP18.1在叶片表皮细胞中的亚细胞定位。

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.

图5 烟草沉默HSP18.1后的表型（A）、相对电导率与基因表达量变化（B）和DAB与NBT染色（C） HSP18.1：基因沉默株；GFP：空载对照；DAB：二氨基联苯胺染色，颜色深表示SOD高；NBT：硝基蓝四氮唑染色，颜色深显示活性氧含量多。* 表示在0.05水平差异显著。

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.

图6 ‘安吉拉’月季愈伤组织RcHSP18.1过表达株（OX）与野生型（WT）的RcHSP18.1表达量和相对电导率 * 表示在0.05水平差异显著，** 表示在0.01水平差异显著。

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.

图7 ‘安吉拉’月季沉默RcHSP18.1后的表型基因（A）、基因表达量（B）和DAB与NBT染色（C） TRV2-HSP18.1：基因沉默株；TRV2-GFP：空载对照；DAB：二氨基联苯胺染色，颜色深表示SOD高；NBT：硝基蓝四氮唑染色，颜色深显示活性氧含量多。*** 表示在0.001水平差异显著。

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.

图8 ‘安吉拉’月季中RcHSF30响应热胁迫的表达及其编码蛋白与HSP18.1互作关系测定 ** 表示在0.01水平差异显著。

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.

参考文献 23

[1]	Chai M L, Xu C J, Senthil K K, Kim D H. 2002. Stable transformation of protocorm-like bodies in Phalaenopsis orchid mediated by Agrobacterium tumefaciens. Scientia Horticulturae, 96 (1-4):213-224.
[2]	Gong Z, Xiong L, Shi H, Li X, Zhao T. 2020. Plant abiotic stress response and nutrient use efficiency. Science China:Life Sciences, 63 (5):40.
[3]	Gu L, Jiang T, Zhang C, Li X, Wang C, Zhang Y, Li T, Dirk L M A, Downie A B, Zhao T. 2019. Maize HSFA2 and HSBP2 antagonistically modulate raffinose biosynthesis and heat tolerance in Arabidopsis. The Plant Journal, 100 (1):128-142.
[4]	Guo Yan,Dong Chenghu,Bai Yujia,Feng Zuoshan. 2012. Research on the wax extraction process of Rosa chinensis. Preservation and Processing, 12 (3):34-37. (in Chinese)
	郭焰, 董成虎, 白羽嘉, 冯作山. 2012. 玫瑰花蜡质提取工艺的研究. 保鲜与加工, 12 (3):34-37.
[5]	Hahn A, Bublak D, Schleiff E, Scharf K D. 2011. Crosstalk between Hsp90 and Hsp70 chaperones and heat stress transcription factors in tomato. The Plant Cell, 23 (2):741-755.
[6]	Hua Zhirui, Li Xiaoling. 2009. Advances in resistance physiology and breeding of lilies. Shaanxi Agricultural Sciences, 55 (2):106-108. (in Chinese)
	华智锐, 李小玲. 2009. 百合的抗性生理及抗性育种研究进展. 陕西农业科学, 55 (2):106-108.
[7]	Kan Y, Mu X R, Zhang H, Gao J, Shan J X, Ye W W, Lin H X. 2022. TT 2 controls rice thermotolerance through SCT1-dependent alteration of wax biosynthesis. Nature Plants, 8 (1):53-67. doi: 10.1038/s41477-021-01039-0 pmid: 34992240
[8]	Kang Chao, Li Bowen, Yang Binan, Ming Feng. 2022. Research on drought resistance physiology of different Rosa chinensis varieties. Smart Agriculture Guide,(19):24-26. (in Chinese)
	康超, 李博文, 杨碧楠, 明凤. 2022. 不同月季品种抗旱生理研究. 智慧农业导刊, 2 (19):24-26.
[9]	Kong Lingjie, Chen Yanbo, Wang Yaqin. 2019. Studies on heat tolerance of different summer chrysanthemum cultivars. Acta Horticulturae Sinica, 46 (12):2437-2448. (in Chinese) doi: 10.16420/j.issn.0513-353x.2019-0145
	孔令接, 陈言博, 王亚琴. 2019. 不同夏菊品种的耐热性研究. 园艺学报, 46 (12):2437-2448. doi: 10.16420/j.issn.0513-353x.2019-0145
[10]	Li Zheng, Liu Bing, Zhou Hong, Wang Xiuyun, Xia Yiping. 2021. Isolation and function analysis of the promoter of a thermal inducible gene RCA1 in Rhododendron hainanense. Acta Horticulturae Sinica, 48 (3):566-576. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-0483
	李铮, 刘冰, 周泓, 王秀云, 夏宜平. 2021. 海南杜鹃热诱导基因RhRCA1启动子的克隆与功能分析. 园艺学报, 48 (3):566-576. doi: 10.16420/j.issn.0513-353x.2020-0483
[11]	Liu G T, Chai F M, Wang Y, Jiang J Z, Duan W, Wang Y T, Wang F F, Li S H, Wang L J. 2018. Genome-wide identification and classification of HSF family in grape,and their transcriptional analysis under heat acclimation and heat stress. Horticultural Plant Journal, 4 (4):133-143.
[12]	Peng Yongzheng, Liu Zhiyuan, Zhu Xiaofei, Du Xiwu, Ye Kang, Lu Yanqing, Qin Jun, Zeng Li. 2019. Physiological index changes and heat tolerance evaluation of five Rosa chinensis varieties after high temperature treatment. Journal of Shanghai Jiaotong University(Agricultural Science Edition), 37 (5):53-58. (in Chinese)
	彭勇政, 刘智媛, 朱晓非, 杜习武, 叶康, 陆燕青, 秦俊, 曾丽. 2019. 5个月季品种高温处理后生理指标变化及其耐热性评价. 上海交通大学学报(农业科学版), 37 (5):53-58.
[13]	Shah K, Nahakpam S. 2012. Heat exposure alters the expression of SOD,POD,APX and CAT isozymes and mitigates low cadmium toxicity in seedlings of sensitive and tolerant rice cultivars. Plant Physiology and Biochemistry, 57:106-113.
[14]	Sun Z, Kumar R M S, Li J, Yang G, Xie Y. 2022. In Silico search and biological validation of MicroR171 family related to abiotic stress response in mulberry(Morus alba). Horticultural Plant Journal, 8 (2):184-194.
[15]	Tian Yujing, Yin Xueren, Li Xian, Chen Kunsong. 2017. Regulation of stress responses by heat stress transcription factors(Hsfs)in plants. Acta Horticulturae Sinica, 44 (1):179-192. (in Chinese) doi: 10.16420/j.issn.0513-353x.2016-0570
	田尉婧, 殷学仁, 李鲜, 陈昆松. 2017. 热激转录因子调控植物逆境响应研究进展. 园艺学报, 44 (1):179-192. doi: 10.16420/j.issn.0513-353x.2016-0570
[16]	Wang Chunyu, Li Zhengjun, Wang Ping, Zhang Lixia. 2023. Physiological and biochemical analysis of the drought resistance of the Sorghum bicolor sb1 mutant with cuticular wax deficiency. Biotechnology Bulletin, 39 (5):160-167. (in Chinese) doi: 10.13560/j.cnki.biotech.bull.1985.2022-0889
	王春语, 李政君, 王平, 张丽霞. 2023. 高粱表皮蜡质缺失突变体sb1抗旱生理生化分析. 生物技术通报, 39 (5):160-167. doi: 10.13560/j.cnki.biotech.bull.1985.2022-0889
[17]	Wang Jingwen, Feng Bin, Chen Xiyu. 2022. Highway potted Rosa chinensis cultivation techniques for large-scale maintenance on elevated structures. Modern Horticulture, 45 (5):73-75. (in Chinese)
	王靖文, 冯彬, 陈昕宇. 2022. 高架月季盆栽规模化养护技术. 现代园艺, 45 (5):73-75.
[18]	Wang Tao,Tian Xueyao,Xie Yanfeng,Zhang Wangxiang. 2013. Advancements in the study of plant heat tolerance. Journal of Yunnan Agricultural University(Natural Science), 28 (5):719-726. (in Chinese)
	王涛, 田雪瑶, 谢寅峰, 张往祥. 2013. 植物耐热性研究进展. 云南农业大学学报(自然科学), 28 (5):719-726.
[19]	Wu Z, Li T, Xiang J, Teng R, Zhang D, Teng N. 2023. A lily membrane-associated NAC transcription factor LlNAC014 is involved in thermotolerance via activation of the DREB2-HSFA3 module. J Exp Bot, 74 (3):945-963.
[20]	Zhao D Q, Xia X, Su J H, Wei M G, Wu Y Q, Tao J. 2019. Overexpression of herbaceous peony HSP 70 confers high temperature tolerance. BMC Genomics, 20:70.
[21]	Zhao Xi,Zhang Tingting,Xing Wenting,Wang Jian,Song Xiqiang,Zhou Yang. 2021. Genome-wide identification and expression analysis under temperature stress of HSP 70 gene family in Dendrobium catenatum. Acta Horticulturae Sinica, 48 (9):1743-1754. (in Chinese)
	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家族基因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
[22]	Zhang Fangjing. 2019. Functional validation of HSF(Heat Shock Factor)transcription factors in Rosa chinensis[M.D. Dissertation]. Changsha:Central South University of Forestry and Technology. (in Chinese)
	张方静. 2019. 月季HSF转录因子的功能验证[硕士论文]. 长沙: 中南林业科技大学.
[23]	Zhou Y, Wang X, Qi K, Bao J, Zhang S, Gu C. 2023. Involvement of long non-coding RNAs in pear fruit senescence under high- and low-temperature conditions. Horticultural Plant Journal, 9 (2):224-236.