Gene Cloning，Expression Analysis，and Subcellular Localization of ChXES1 and ChXES2 in Calibrachoa hybrida

doi:10.15933/j.cnki.1004-3268.2025.08.012

Abstract

Abstract: Identification and cloning of the xanthophyll esterase gene XES from Calibrachoa hybrida，as well as investigation of its expression characteristics in different tissues and under stress conditions，and its subcellular localization，will lay the foundation for exploring the functions of the XES gene. Based on the transcriptome and genome databases of C.hybrida cv.Fengbaohuang，ChXES genes and promoters were identified and analyzed by DNAMAN and other softwares，and cloned using PCR technology.RT‑qPCR was used to analyze the expression patterns of ChXESs in different tissues，flower developmental stages，and leaves under abiotic stress，and the subcellular localization was observed by injecting Agrobacterium into tobacco.The findings indicated that two ChXES genes were identified in C.hybrida，both encoding 710 amino acids and featuring ABH and LPLAT domains.Phylogenetic analysis revealed their closest relationship with Petunia hybrida.The promoter regions of ChXES1pro and ChXES2pro contained stress‑responsive and hormone‑responsive elements.ChXESs were expressed in all tested tissues. During flower development，carotenoid content increased，with ChXES1 showing higher expression levels than ChXES2.ChXES1 responded more significantly to cold and drought stress，reaching its highest transcription levels at 12 h and 6 h，respectively.In contrast，ChXES2 exhibited a stronger response to heat stress and ABA signaling，with peak expression at 18 h for both conditions.ChXES1 and ChXES2 were preliminarily localized to the chloroplast and plastoglobules.ChXES1 and ChXES2 demonstrate tissue‑specific expression in C.hybrida，and their response patterns to stress and ABA are different，which provides guidance for further study on the functions of ChXES1 and ChXES2，regulation of flower color and resistance mechanism.

Key words: Calibrachoa hybrida, Xanthophyll esterase gene, Promoter, Expression analysis, Abiotic stress, Subcellular localization

摘要： 对舞春花叶黄素酯酶基因XES进行鉴定与克隆，研究其在不同组织和逆境下的表达特征以及亚细胞定位，为挖掘XES基因功能奠定基础。基于舞春花风暴黄转录组和基因组数据库，使用DNAMAN等软件及PCR技术对ChXESs基因和启动子进行鉴定、分析及克隆，利用RT-qPCR技术检测ChXESs在不同组织、花发育阶段及非生物胁迫后叶片的表达变化，通过农杆菌注射烟草观察亚细胞定位情况。结果表明，在舞春花中鉴定到2个ChXES基因，均编码710个氨基酸，具有ABH和LPLAT结构域，与矮牵牛亲缘关系最近，ChXES1pro和ChXES2pro含有逆境和激素响应元件。ChXESs 在不同组织均有表达，在花发育中，类胡萝卜素含量增加，ChXES1 表达量高于ChXES2；ChXES1对低温和干旱胁迫响应更显著，分别在12 h和6 h转录水平最高；ChXES2对高温胁迫和ABA信号响应更剧烈，均在18 h表达量达到峰值。ChXES1和ChXES2亚细胞初步定位于叶绿体和质体小球。ChXES1和ChXES2在舞春花组织中特异性表达，对逆境和ABA响应模式不同，为深入研究ChXES1和ChXES2功能、舞春花花色调控和抗逆机制提供指导。

关键词: 舞春花, 叶黄素酯酶基因, 启动子, 表达分析, 非生物胁迫, 亚细胞定位

CLC Number:

S682

SONG Junyan, ZHANG Yuanshan, SHEN Xiaohui, ZHAO Ying, CHEN Guanqun. Gene Cloning，Expression Analysis，and Subcellular Localization of ChXES1 and ChXES2 in Calibrachoa hybrida[J]. Journal of Henan Agricultural Sciences, 2025, 54(8): 122-132.

宋军燕, 张源珊, 申晓辉, 赵莹, 陈冠群. 舞春花ChXES1和ChXES2基因克隆、表达分析及亚细胞定位[J]. 河南农业科学, 2025, 54(8): 122-132.

Figures/Tables 9

References

［1］LIU G F，SUN M M，ZOU P S，et al.The complete chloroplast genome sequence of a popular ornamental plant Calibrachoa hybrida（Solanaceae：Petunioideae）［J］.Mitochondrial DNA Part B，2020，5（3）：3374‑3375.

［2］KISHIMOTO S，ODA‑YAMAMIZO C，OHMIYA A.Comparison of Petunia and Calibrachoa in carotenoid pigmentation of corollas［J］. Breeding Science，2019，69 （1）：117‑126.

［3］王跃兵. 园林新优景观植物：小花矮牵牛盆栽技术探析［J］.现代园艺，2019（1）：58‑59.

WANG Y B. Analysis on potting techniques of Petunia parviflora，a new excellent landscape plant in garden［J］.Contemporary Horticulture，2019（1）：58‑59.

［4］KISHIMOTO S，ODA‑YAMAMIZO C，OHMIYA A.Regulation of carotenoid pigmentation in corollas of Petunia［J］. Plant Molecular Biology Reporter，2018，36 （4）：632‑642.

［5］WATKINS J L，LI M，MCQUINN R P，et al. A GDSL esterase/lipase catalyzes the esterification of lutein in bread wheat［J］. The Plant Cell，2019，31（12）：3092‑3112.

［6］汪书杰，栾雨婷，徐昌杰. 植物叶黄素酯化研究进展［J］.园艺学报，2023，50（9）：1830‑1840.
WANG S J，LUAN Y T，XU C J. Research progresses on plant xanthophyll esterification［J］.Acta Horticulturae Sinica，2023，50（9）：1830‑1840.

［7］LIPPOLD F，VOM DORP K，ABRAHAM M，et al. Fatty acid phytyl ester synthesis in chloroplasts of Arabidopsis［J］.The Plant Cell，2012，24（5）：2001‑2014.

［8］ARIIZUMI T，KISHIMOTO S，KAKAMI R，et al.Identification of the carotenoid modifying gene pale yellow petal 1 as an essential factor in xanthophyll esterification and yellow flower pigmentation in tomato（Solanum lycopersicum）［J］. The Plant Journal，2014，79 （3）：453‑465.

［9］KISHIMOTO S，ODA‑YAMAMIZO C，OHMIYA A.Heterologous expression of xanthophyll esterase genes affects carotenoid accumulation in Petunia corollas［J］.Scientific Reports，2020，10：1299.

［10］IIJIMA L，KISHIMOTO S，OHMIYA A，et al. Esterified carotenoids are synthesized in petals of carnation（Dianthus caryophyllus）and accumulate in differentiated chromoplasts［J］. Scientific Reports，2020，10（1）：15256.

［11］LI R H，ZENG Q Y，ZHANG X X，et al.Xanthophyll esterases in association with fibrillins control the stable storage of carotenoids in yellow flowers of rapeseed（Brassica juncea）［J］. New Phytologist，2023，240（1）：285‑301.

［12］GUAN Z L，LI X W，YANG J S，et al. The mechanism of white flower formation in Brassica rapa is distinct from that in other Brassica species［J］.Theoretical and Applied Genetics，2023，136（6）：133.

［13］LI P R，LV S R，ZHANG D S，et al.The carotenoid esterification gene BrPYP controls pale‑yellow petal color in flowering Chinese cabbage（Brassica rapa L.subsp.parachinensis）［J］. Frontiers in Plant Science，2022，13：844140.

［14］BERRY H M，RICKETT D V，BAXTER C J，et al.Carotenoid biosynthesis and sequestration in red chilli pepper fruit and its impact on colour intensity traits［J］.Journal of Experimental Botany，2019，70（10）：2637‑2650.

［15］ZACARÍAS‑GARCÍA J，LUX P E，CARLE R，et al.Characterization of the Pale Yellow Petal/Xanthophyll Esterase gene family in Citrus as candidates for carotenoid esterification in fruits［J］.Food Chemistry，2021，342：128322.
［16］袁美静，马誉，巫瑞，等. 影响月季花瓣呈色的理化因子及花色苷组分分析［J］.西北植物学报，2024，44（2）：255‑269.

YUAN M J，MA Y，WU R，et al. Physicochemical characteristics and anthocyanin components affecting the color of rose petals［J］.Acta Botanica Boreali‑Occidentalia Sinica，2024，44（2）：255‑269.

［17］CHEN G Q，HE W Z，GUO X X，et al.Genome‑wide identification，classification and expression analysis of the MYB transcription factor family in Petunia［J］.International Journal of Molecular Sciences，2021，22 （9）：4838.

［18］SUN T H，RAO S，ZHOU X S，et al. Plant carotenoids：Recent advances and future perspectives［J］.Molecular Horticulture，2022，2（1）：3.

［19］段敏杰，杨小苗，李怡斐，等. 辣椒MADS-box转录因子CaRIN基因克隆、表达与功能分析［J］. 西北植物学报，2024，44（8）：1239‑1249.
DUAN M J，YANG X M，LI Y F，et al. Cloning，expression，and functional analysis of the CaRIN gene，encoding a MADS‑box transcription factor from pepper［J］. Acta Botanica Boreali‑Occidentalia Sinica，2024，44（8）：1239‑1249.

［20］SUN Q，HE Z C，WEI R R，et al.The transcriptional regulatory module CsHB5‑CsbZIP44 positively regulates abscisic acid‑mediated carotenoid biosynthesis in Citrus（Citrus spp.）［J］. Plant Biotechnology Journal，2024，22（3）：722‑737.

［21］JIA D J，LI Y C，JIA K，et al. Abscisic acid activates transcription factor module MdABI5-MdMYBS1 during carotenoid‑derived apple fruit coloration［J］.Plant Physiology，2024，195（3）：2053‑2072.

［22］YANG Y Z，LI T，TENG R M，et al. Low temperature effects on carotenoids biosynthesis in the leaves of green and albino tea plant（Camellia sinensis（L.）O.Kuntze）［J］. Scientia Horticulturae，2021，285：110164.

［23］梁婉凤，曾菁菁，胡若群，等. 转录组与代谢组分析不同生长时期金线莲类胡萝卜素的积累［J］. 生物技术通报，2024，40（10）：262‑274.
LIANG W F，ZENG J J，HU R Q，et al. Transcriptional and metabolomic analysis of carotenoid accumulation in Anoectochilus roxburghii during different growth periods［J］.Biotechnology Bulletin，2024，40（10）：262‑274.
［24］陈雯新，李萌，孙悦宸，等. 茄子衰老叶质体小球降解途径的观察［J］.中国瓜菜，2021，34（5）：109‑112.
CHEN W X，LI M，SUN Y C，et al. Observation on the degradation pathways of plastoglobules in senescent eggplant leaves［J］. China Cucurbits and Vegetables，2021，34（5）：109‑112.
［25］ROTTET S，DEVILLERS J，GLAUSER G，et al.Identification of plastoglobules as a site of carotenoid cleavage［J］. Frontiers in Plant Science，2016，7：1855.

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