月季RhRNF185-like的克隆及其对花瓣衰老影响分析

doi:10.16420/j.issn.0513-353x.2023-0373

摘要/Abstract

摘要：

为探究月季（Rosa hybrida）花瓣衰老机制以及为通过分子育种等手段创制抗衰老品种提供基因储备，以切花月季‘卡罗拉’（Carola）为试材，通过月季转录组测序筛选到1个E3泛素连接酶RING结构域家族基因RhRNF185-like。qRT-PCR结果表明，在花朵开放的中后期3 ~ 5级的花瓣中RhRNF185-like的表达量显著增强，说明其可能影响了月季花瓣的衰老。将其在烟草叶肉细胞进行瞬时过表达，通过激光共聚焦扫描显微镜观察到RhRNF185-like的GFP信号与DAPI核信号重合，说明其定位在细胞核上。利用病毒诱导的基因沉默技术将RhRNF185-like在月季花瓣中沉默，发现沉默的花瓣圆片衰老程度明显大于对照。此外，与对照相比，RhRNF185-like沉默的花瓣圆片中衰老标志基因RhSAG12的相对表达量、离子渗透率和褪色率显著升高，均说明沉默RhRNF185-like促进了月季花瓣的衰老。以上结果表明，RhRNF185-like以拮抗调节的方式影响了月季花瓣衰老。

关键词: 月季, 花瓣, 衰老, RhRNF185-like

Abstract:

In order to explore the senescence mechanism of rose petals and provide gene reserve for the creation of anti-senescence rose varieties through molecular breeding and other means，a RhRNF185-like gene belonging to E3 ubiquitin ligase RING domain family were screened by transcriptome sequencing using cut rose‘Carola’as the test material in this study. The results of qRT-PCR showed that the expression level of RhRNF185-like in the rose petals was significantly increased in the middle and late stage of rose flower opening，indicating that RhRNF185-like expression might affect the senescence of rose petals. Transient overexpression of RhRNF185-like in tobacco mesophyll cells showed that the GFP signal of RhRNF185-like coincided with the nuclear signal of DAPI through Confocal microscope，indicating that RhRNF185-like was located in the nucleus. Virus-induced gene silencing technique（VIGS）was used to silence RhRNF185-like gene in rose petals. It was found that the senescence degree of petals with RhRNF185-like gene silencing was significantly greater than that of TRV control group. In addition，compared with TRV control group，the relative expression level of senescence marker gene RhSAG12，ion permeability and fading rate of rose petals were significantly increased in the RhRNF185-like gene silencing group，indicating that silencing RhRNF185-like promoted the senescence of rose petals. These results indicated that RhRNF185-like affected the senescence of rose petals in an antagonistic regulatory manner.

Key words: rose, petal, senescence, RhRNF185-like

王雯雨, 王家荫, 杜婷婷, 张晶晶, 张超, 辛翠花, 郭江波, 裴海霞. 月季RhRNF185-like的克隆及其对花瓣衰老影响分析[J]. 园艺学报, 2024, 51(5): 1047-1055.

WANG Wenyu, WANG Jiayin, DU Tingting, ZHANG Jingjing, ZHANG Chao, XIN Cuihua, GUO Jiangbo, PEI Haixia. Cloning of RhRNF185-like Gene from Rose and Its Effect on Petal Senescence[J]. Acta Horticulturae Sinica, 2024, 51(5): 1047-1055.

图/表 7

表1 试验中所用引物序列

Table 1 The sequences of primers used for experiment

引物名称 Primer name	引物序列（5′-3′） Primer sequence
RhUBI2-qPCR	F：GCCCTGGTGCGTTCCCAACTG；R：CCTGCGTGTCTGTCCGCATTG
RhRNF185-like-qPCR	F：CAATGACGCAGGGGATTTCG；R：ATGTGAGTGATGGTGGAGCC
RhRNF185-like-VIGS	F：GTTTTGGGGAATCAACGAGC；R：TCAGCGCAGCAATCACAAAT
RhRNF185-like-ORF	F：ATGGCAAGTGGTTTTGGGGAAT；R：CTAACAAAAAATCAGCGCAGC
SAG12-qPCR	F：AAGACCAATCCAAAAGCAAC；R：AATCAAAACCACCTCCTTCA

图1 不同开放等级月季花瓣中RhRNF185-like的相对表达量

Fig. 1 Relative expression of RhRNF185-like in different opening grades of Chinese rose * P < 0.05，** P < 0.01.

图2 RhRNF185-like的ORF克隆（A）及利用沉默载体pYY13、过表达定位载体pJG186筛选鉴定（B、C）

Fig. 2 ORF cloning of RhRNF185-like（A）and screening and identification of silencing vector pYY13 and overexpression localization vector pJG186（B，C）

图3 RhRNF185-like系统发育树分析 FV：草莓；Md：苹果；Mn：川桑；Pp：桃；Pm：梅花；Me：木薯。

Fig. 3 Phylogenetic tree analysis of RhRNF185-like FV：Fragaria vesca subsp.；Md：Malus × domestica；Mn：Morus notabilis；Pp：Prunus persica；Pm：Prunus mume；Me：Manihot esculenta.

图4 RhRNF185-like亚细胞定位

Fig. 4 RhRNF185-like subcellular localization

图5 月季花瓣圆片RhRNF185-like沉默后的表型和该基因与衰老标志基因RhSAG12的表达量

Fig. 5 Phenotype of rose petal disc RhRNF185-like silencing and relative expression of this gene and senescence marker gene RhSAG12 ** P < 0.01.

图6 月季花瓣圆片RhRNF185-like沉默后的离子渗透率和褪色率

Fig. 6 Ion permeability and color fading rate of rose petal disc RhRNF185-like after silencing * P < 0.05，** P < 0.01.

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