月季‘月月红’RcTCP9基因在盐和干旱胁迫中的功能分析

doi:10.16420/j.issn.0513-353x.2024-0791

摘要/Abstract

摘要：

以月季‘月月红’（Rosa chinensis‘Yueyuehong’）扦插苗为材料，分析了RcTCP9基因在盐和干旱胁迫不同时间的表达模式，结果表明，在盐和干旱胁迫条件下RcTCP9的表达量显著上调，说明其对两种胁迫有积极响应。将RcTCP9在烟草中瞬时表达，通过观察绿色荧光信号发现其编码蛋白定位于细胞核上。病毒诱导的基因沉默（VIGS）技术和瞬时过表达试验发现，RcTCP9的沉默会导致植株在盐和干旱胁迫下出现叶片及根系萎蔫等生理损伤表征，植物细胞离子渗透率及丙二醛（MDA）含量增加，过氧化氢酶（CAT）及过氧化物酶（POD）活性水平则降低。过表达的月季花瓣应对两种胁迫细胞膜损伤减少，褪色率和MDA含量降低，表现出更好的抗性。测定过表达植株在盐和干旱胁迫下耐逆相关基因的表达水平，结果显示RcTCP9的表达可能通过调控RcPR4-1和RcNAC091相关通路来增强植物的胁迫耐受性。综上所述，RcTCP9在提高月季对盐和干旱胁迫的耐受性方面具有重要作用。

关键词: 月季, RcTCP9, 耐盐性, 耐旱性, 抗氧化

Abstract:

Using cuttings of Rosa chinensis‘Yueyuehong’as materials，the expression patterns were analyzed of the RcTCP9 gene under varying durations of salt and drought stress. Results indicated that the expression of RcTCP9 was significantly upregulated under both stress conditions，demonstrating its active response. The gene was transiently expressed in tobacco，and observations of green fluorescence signals revealed that the encoded protein localized to the nucleus. Using Virus-Induced Gene Silencing（VIGS）and transient overexpression experiments，silencing RcTCP9 led to physiological damage in plants under salt and drought stress，characterized by wilting of leaves and roots，increased ion permeability，and elevated malondialdehyde（MDA）levels，while the activities of catalase（CAT）and peroxidase（POD）decreased. Overexpressed rose petals exhibited reduced cellular membrane damage，lower fading rates，and decreased MDA levels，showing improved resistance. The expression levels of stress tolerance related genes in overexpressed plants under salt and drought stress were determined，and the results showed that the expression of RcTCP9 may enhance plant stress tolerance by regulating the RcPR4-1 and RcNAC091 related pathways. In summary，the RcTCP9 gene plays a significant role in enhancing the tolerance of roses to salt and drought stress.

Key words: rosa, RcTCP9, salt tolerance, drought tolerance, antioxidant

董炫克, 李映旸, 马豫皖, 蔡艳芳, 丁启涵, 陈海霞, 李玉帆, 陈己任. 月季‘月月红’RcTCP9基因在盐和干旱胁迫中的功能分析[J]. 园艺学报, 2025, 52(7): 1789-1802.

DONG Xuanke, LI Yingyang, MA Yuwan, CAI Yanfang, DING Qihan, CHEN Haixia, LI Yufan, and CHEN Jiren. The Functional Analysis of RcTCP9 Gene in Response to Salt and Drought Stress in Rosa chinensis‘Yueyuehong’[J]. Acta Horticulturae Sinica, 2025, 52(7): 1789-1802.

图/表 12

表1 试验中所用引物序列

Table 1 The sequences of primers used for experiment

用途 Purpose	名称 Name	引物序列（5′-3′） Primer sequence
克隆RcTCP9基因CDS全长 Clone the full-length CDS of the RcTCP9 gene	KL-RcTCP9-F	ATGACGTCGTATTTTGAGGATCAAG
	KL-RcTCP9-R	TCACTGGCTTCCTGGGCCTG
亚细胞定位载体构建 Construction of subcellular localization vector	pBI121-RcTCP9-F	ACGGGGGACTCTAGAGGATCCATGACGTCGTATTTTGAGGA
亚细胞定位载体构建 Construction of subcellular localization vector	pBI121-RcTCP9-R	GGACTGACCACCCGGGGATCCCTGGCTTCCTGGGCCTGCGG
瞬时过表达载体构建 Construction of transient overexpression vector	1305-RcTCP9-F	GGAGAGAACACGGGGGACATGACGTCGTATTTTGAGGATCA
瞬时过表达载体构建 Construction of transient overexpression vector	1305-RcTCP9-R	CATCATGGTCTTTGTAGTCCTGGCTTCCTGGGCCTGCGGCGGG
VIGS基因沉默载体构建 Construction of VIGS gene silencing vector	TRV2-RcTCP9-F	CTGTGAGTAAGGTTACCGAATTCGTTCGTGGGGAACGGCGGAG
VIGS基因沉默载体构建 Construction of VIGS gene silencing vector	TRV2-RcTCP9-R	CGCGTGAGCTCGGTACCGGATCCCTTCCTGGGCCTGCGGCGG
实时荧光定量表达（qRT-PCR）	RcTCP9-F	TGGTAGTGGTAACAGTAACA
	RcTCP9-R	CTCCTGCTTATCGTAAATCTC
	RcCTP-F	GGGTGATGATGCAGCTTT
	RcCTP-R	TTAGCACTTGACCTCCTTCA
	RcPR4/1-F	ATGGCCGGAAAACAATGC
	RcPR4/1-R	GGGCTTGTCGGCATCC
	RcNAC091-F	TGTCTTCCTCGGAGTTACAGTTACC
	RcNAC091-R	CCCAGGGGTCGTATTTGTACAG

图1 RcTCP9的CDS序列克隆

Fig. 1 The cloning of the CDS sequence of RcTCP9

图2 TRV2-RcTCP9沉默载体的构建 A：RcTCP9有效片段与TRV2-RcTCP9比对结果；B：菌落PCR凝胶电泳图

Fig. 2 Construction of the TRV2-RcTCP9 silencing vector A：Comparison results between the effective fragment of RcTCP9 and TRV2-RcTCP9；B：Gel electrophoresis image of colony PCR

图3 月季RcTCP9与其他物种TCP蛋白的的系统进化树

Fig. 3 Phylogenetic tree of RcTCP9 from Rosa chinensis and TCP proteins of other species

图4 RcTCP9在烟草叶片中的亚细胞定位

Fig. 4 The subcellular localization of RcTCP9 in tobacco leaves

图5 月季‘月月红’扦插苗在盐和干旱胁迫下RcTCP9的组织表达分析 *P < 0.05，**P < 0.01。下同

Fig. 5 Tissue-specific expression analysis of RcTCP9 in cuttings of Rosa chinensis‘Yueyuehong’under salt and drought stress *P < 0.05，**P < 0.01. The same below

图6 盐胁迫下VIGS沉默RcTCP9组和对照组月季扦插苗表型、RcTCP9表达量及生理指标变化

Fig. 6 The changes of phenotype phenotype，RcTCP9 expression and physiological indexes of roses cutting seedlings in VIGS silencing RcTCP9 group and control group under salt stress

图7 盐胁迫下月季抗氧化酶活性相关指标

Fig. 7 Antioxidant enzyme activity related indexes of roses under salt stress

图8 盐胁迫下过表达RcTCP9对月季耐盐性的影响 EV：空载组；OE：过表达

Fig. 8 Overexpression of RcTCP9 enhanced the salt tolerance of roses EV：Empty vector；OE：Overexpression

图9 干旱胁迫下VIGS沉默RcTCP9组和对照组月季扦插苗表型、RcTCP9表达量及生理指标变化

Fig. 9 The changes of phenotype，RcTCP9 expression and physiological indexes of roses cuttings in VIGS silencing RcTCP9 group and control group under drought stress

图10 干旱胁迫下月季抗氧化酶活性相关指标

Fig. 10 Antioxidant enzyme activity related indexes of rose under drought stress

图11 干旱胁迫下过表达RcTCP9对月季耐旱性的影响

Fig. 11 Overexpression of RcTCP9 enhanced the drought tolerance of roses

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