月季Harbinger类转座子基因RcHTD的克隆与功能初探

doi:10.16420/j.issn.0513-353x.2024-0842

园艺学报 ›› 2025, Vol. 52 ›› Issue (9): 2377-2386.doi: 10.16420/j.issn.0513-353x.2024-0842

• 遗传育种·种质资源·分子生物学 • 上一篇下一篇

月季Harbinger类转座子基因RcHTD的克隆与功能初探

李茂福¹^,²^,³, 杨媛¹^,²^,⁴, 王华¹^,²^,³, 孙佩¹^,²^,³, 康岩慧¹^,²^,³, 孙向一¹^,²^,³, 罗洁¹^,²^,³, 刘欣¹^,²^,³, 金敏¹^,²^,³, 金万梅¹^,²^,³^,^*()

¹ 北京市农林科学院林业果树研究所，北京 100093
² 农业部华北地区园艺作物生物学与种质创制重点实验室，北京 100093
³ 北京市功能花卉工程技术研究中心，北京 100093
⁴ 北京市落叶果树工程技术研究中心，北京 100093

收稿日期:2025-01-05 修回日期:2025-04-07 出版日期:2025-09-25 发布日期:2025-09-24
通讯作者:
^* E-mail：jwm0809@163.com
基金资助:
国家自然科学基金面上项目(32172608); 北京市农林科学院科技创新能力专项(KJCX20230110); 北京市农林科学院科技创新能力专项(KJCX20230221)

Cloning and Preliminary Functional Exploration of the Transposon-Derived Gene RcHTD in Rose

LI Maofu¹^,²^,³, YANG Yuan¹^,²^,⁴, WANG Hua¹^,²^,³, SUN Pei¹^,²^,³, KANG Yanhui¹^,²^,³, SUN Xiangyi¹^,²^,³, LUO Jie¹^,²^,³, LIU Xin¹^,²^,³, JIN Min¹^,²^,³, JIN Wanmei¹^,²^,³^,^*()

¹ Institute of Forestry and Pomology， Beijing Academy of Agriculture and Forestry Sciences， Beijing 100093， China
² Key Laboratory of Biology and Genetic Improvement of Horticultural Crops（North China）， Ministry of Agriculture， Beijing 100093， China
³ Beijing Engineering Research Center of Functional Floriculture， Beijing 100093， China
⁴ Beijing Engineering Research Center for Deciduous Fruit Trees， Beijing 100093， China

Received:2025-01-05 Revised:2025-04-07 Published:2025-09-25 Online:2025-09-24

摘要/Abstract

摘要：

为研究月季Harbinger类转座子基因RcHTD（Harbinger transposase-derived）在花青素合成中的调控作用，以‘月月红’（Rosa chinensis‘Slater’s Crimson China’）月季花瓣为材料，前期以Rosa1为诱饵筛选花瓣cDNA文库，获得1个互作基因。本研究中通过克隆获得该基因并命名为RcHTD，其开放阅读框为1 560 bp，编码519个氨基酸。蛋白序列比对发现RcHTD含有DDE转座子亚家族结构域，系统进化树分析表明，RcHTD与编码Harbinger类转座酶的AT5G12010、AT4G29780、AtALP1、AtHHP1亲缘关系最近。通过对RcHTD在‘月月红’花瓣不同发育时期及不同组织中的特异性表达分析发现，其在所有花发育时期及组织中均有表达。亚细胞定位显示RcHTD定位在细胞核中。酵母双杂交证实RcHTD能与RcMYB114直接发生相互作用，凝胶迁移试验显示RcHTD能结合RcMYB114启动子，推测其进一步调控花青素的合成，从而影响月季红色花瓣的形成。

关键词: 月季, 转座子衍生基因, RcHTD, 基因功能

Abstract:

To investigate the regulatory role of Harbinger transposon-derived genes in anthocyanin synthesis in rose，the petals of the rose variety（Rosa chinensis‘Slater’s Crimson China’）were used as experimental material. In preliminary experiments，Rosa1 was employed as bait to screen a petal cDNA library，resulting in the identification of an interacting gene. This study successfully cloned the gene and characterized it as a transposon-derived gene，named RcHTD，which has an open reading frame of 1 560 bp and encodes 519 amino acids. Multiple sequence alignment analysis revealed that RcHTD encompasses a DDE transposon subfamily domain. Phylogenetic tree analysis indicated that RcHTD clustered closely with the genes AT5G12010，AT4G29780，AtALP1 and AtHHP1，which encode Harbinger-type transposases. Expression profilingof RcHTD in different developmental stages and tissues of Rosa chinensis‘Slater’s Crimson China’petals showed that RcHTD is expressed in all stages and tissues. Subcellular localization studies confirmed that the RcHTD protein is localized in the cell nucleus. Yeast two-hybrid experiments indicated that RcHTD physically interacts with RcMYB114. EMSA experiments further confirmed that RcHTD specifically binds to the RcMYB114 promoter，indicating its role in regulating anthocyanin synthesis，thereby consequently influencing the coloration of rose red petal.

Key words: Rosa chinensis, transposon-derived genes, RcHTD, gene function

李茂福, 杨媛, 王华, 孙佩, 康岩慧, 孙向一, 罗洁, 刘欣, 金敏, 金万梅. 月季Harbinger类转座子基因RcHTD的克隆与功能初探[J]. 园艺学报, 2025, 52(9): 2377-2386.

LI Maofu, YANG Yuan, WANG Hua, SUN Pei, KANG Yanhui, SUN Xiangyi, LUO Jie, LIU Xin, JIN Min, JIN Wanmei. Cloning and Preliminary Functional Exploration of the Transposon-Derived Gene RcHTD in Rose[J]. Acta Horticulturae Sinica, 2025, 52(9): 2377-2386.

图/表 8

表1 本研究中所用引物序列

Table 1 Primer sequence used in this study

目的Purpose	引物名称Primer name	引物序列（5′-3′）Primer sequence
基因克隆 Gene cloning	RcHTD	F：ATGGAAGTCACCCCTTTCACATTTCT
基因克隆 Gene cloning	RcHTD	R：CTAATGAAATCCAGTGCCTGCAAGGCC
RT-PCR	RcHTD	F：GCGGAGGAAGGTCGATGAGGACGATACC
	RcHTD	R：CCTGTAATTCGCGTCAAGAGTCTCC
	Actin	F：GGCTGTTCTCTCTCTGTATGC
	Actin	R：TTCTGGGCACCTGAATCTC
亚细胞定位 Subcellular localization	RcHTD	F： CGACTCTAGTCTAGAAAGCTATGGAAGTCACCCCTTTCACATTTCT
亚细胞定位 Subcellular localization	RcHTD	R： CATGGTACCGGATCCACTAGTATGAAATCCAGTGCCTGCAAGGCC
酵母双杂交 Yeast two hybrid	RcHTD	F： TGGCCATGGAGGCCAGTGAATTCATGGAAGTCACCCCTTTCACATTTCT
酵母双杂交 Yeast two hybrid	RcHTD	R： CTACGATTCATCTGCAGCTCGAGCTAATGAAATCCAGTGCCTGCAAGGCC
蛋白诱导表达 Protein induced expression EMAS 探针 EMAS probe	RcHTD Rosa1	F： GATCTGGTTCCGCGTGGATCCATGGAAGTCACCCCTTTCACATTTCT
蛋白诱导表达 Protein induced expression EMAS 探针 EMAS probe	RcHTD Rosa1	R： GATGCGGCCGCTCGAGTCGAATGAAATCCAGTGCCTGCAAGGCC AAATGAATGCTTACTTTCGGAAGTCCTTGTTC AAAAAAAAAAAAAAAATCGGAAGTCCTTGTTC

图1 月季RcHTD 基因的克隆

Fig. 1 Cloning of RcHTD gene from Rosa chinensis

图2 月季RcHTD与拟南芥（AT）蛋白比对及其结构域

Fig. 2 RcHTD protein alignment of Rosa chinensis and Arabidopsis thaliana and its structural domains

图3 月季RcHTD与拟南芥（AT）蛋白进化树分析

Fig. 3 Evolutionary tree analysis of protein from Rosa chinensis RcHTD and Arabidopsis thaliana（AT）

图 4 RcHTD的亚细胞定位

Fig. 4 Subcellular localization analysis of RcHTD

图5 RcHTD在‘月月红’花瓣不同发育时期和不同组织中的表达分析 S1：小蕾期；S2：大蕾期；S3：初开期；S4：盛开期；S5：凋谢期

Fig. 5 Expression analysis of RcHTD gene in different developmental stages and tissues of Rosa chinensis‘Slater’s Crimson China’petals S1：Small bud stage；S2：Big bud stage；S3：Initial opening period；S4：Blooming period；S5：Withering period. t-Test，*P < 0.05

图6 RcHTD 与RcMYB114在酵母中的相互作用

Fig. 6 The interaction between RcHTD and RcMYB114 in yeast

图7 RcHTD 结合Rosa1靶序列重复位点的凝胶迁移分析

Fig. 7 RcHTD binding to the Rosa1 target sequence repeat site by EMSA

参考文献 31

[1]	Bourque G, Burns K H, Gehring M, Gorbunova V, Seluanov A, Hammell M, Imbeault M, Izsvák Z, Levin H L, Macfarlan T S, Mager D L, Feschotte C. 2018. Ten things you should know about transposable elements. Genome Biology, 19 (1):199. doi: 10.1186/s13059-018-1577-z pmid: 30454069
[2]	Bundock P, Hooykaas P. 2005. An Arabidopsis hAT-like transposase is essential for plant development. Nature, 436 (7048):282-284.
[3]	Cam H P, Noma K, Ebina H, Levin H L, Grewal S I. 2008. Host genome surveillance for retrotransposons by transposon-derived proteins. Nature, 451 (7177):431-436.
[4]	Duan C G, Wang X, Xie S, Pan L, Miki D, Tang K, Hsu C C, Lei M, Zhong Y, Hou Y J, Wang Z, Zhang Z, Mangrauthia S K, Xu H, Zhang H, Dilkes B, Tao W A, Zhu J K. 2017. A pair of transposon-derived proteins function in a histone acetyltransferase complex for active DNA demethylation. Cell Research, 27 (2):226-240.
[5]	Fan F, Cheng M, Yuan H, Li N, Liu M, Cai M, Luo X, Ahmad A, Li N, Li S. 2023. A transposon-derived gene family regulates heading date in rice. Plant Science,337:111871.
[6]	Feschotte C, Jiang N, Wessler S R. 2002. Plant transposable elements:where genetics meets genomics. Nature Reviews Genetics, 3 (5):329-341. doi: 10.1038/nrg793 pmid: 11988759
[7]	Gao Y, Xu Z, Zhang L, Li S, Wang S, Yang H, Liu X, Zeng D, Liu Q, Qian Q, Zhang B, Zhou Y. 2020. MYB61 is regulated by GRF4 and promotes nitrogen utilization and biomass production in rice. Nature Communications, 11 (1):5219.
[8]	Hassan A H, Mokhtar M M, El Allali A. 2024. Transposable elements:multifunctional players in the plant genome. Frontiers in Plant Science,14:1330127.
[9]	Jiang N, Bao Z, Zhang X, Eddy S R, Wessler S R. 2004. Pack-MULE transposable elements mediate gene evolution in plants. Nature, 431 (7008):569-573.
[10]	Kapitonov V V, Jurka J. 2004. Harbinger transposons and an ancient HARBI1 gene derived from a transposase. DNA Cell Biology, 23 (5):311-324.
[11]	Krasileva K V. 2019. The role of transposable elements and DNA damage repair mechanisms in gene duplications and gene fusions in plant genomes. Current Opinion in Plant Biology,48:18-25.
[12]	Li M, Zhang H, Yang Y, Wang H, Xue Z, Fan Y, Sun P, Zhang H, Zhang X, Jin W. 2022. Rosa1,a transposable element-like insertion,produces red petal coloration in rose through altering RcMYB114 transcription. Frontiers in Plant Science,13:857684.
[13]	Li Maofu, Yang Yuan, Wang Hua, Liu Jiashen, Jin Wanmei. 2017. Cloning and expression analysis on RcbHLH gene from Rosa chinensis and interaction analysis between MYB and WD 40 transcription factors. Acta Horticulturae Sinica, 44 (10):1949-1958. (in Chinese)
	李茂福, 杨媛, 王华, 刘佳棽, 金万梅. 2017. 月季bHLH基因的克隆、表达及其与MYB和WD40的互作分析. 园艺学报, 44 (10):1949-1958. doi: 10.16420/j.issn.0513-353x.2017-0183
[14]	Liu Keke, Tang Dingqin, Zhou Mingbing. 2021. The evolution pathway of transposon-derived genes and its effect on biological growth and development. Chinese Journal of Cell Biology, 43 (1):152-160. (in Chinese)
	刘珂珂, 汤定钦, 周明兵. 2021. 转座子衍生基因的演变途径及对生物生长发育的影响. 中国细胞生物学学报, 43 (1):152-160
[15]	Mao D, Tao S, Li X, Gao D, Tang M, Liu C, Wu D, Bai L, He Z, Wang X, Yang L, Zhu Y, Zhang D, Zhang W. 2022. The Harbinger transposon-derived gene PANDA epigenetically coordinates panicle number and grain size in rice. Plant Biotechnology Journal, 20 (6):1154-1166.
[16]	Mikina W, Hałakuc P, Milanowski R. 2024. Transposon-derived introns as an element shaping the structure of eukaryotic genomes. Journal of Experimental Botany, 15 (1):15.
[17]	Naito K, Zhang F, Tsukiyama T, Saito H, Hancock C N, Richardson A O, Okumoto Y, Tanisaka T, Wessler S R. 2009. Unexpected consequences of a sudden and massive transposon amplification on rice gene expression. Nature, 461 (7267):1130-1134.
[18]	Niu X M, Xu Y C, Li Z W, Bian Y T, Hou X H, Chen J F, Zou Y P, Jiang J, Wu Q, Ge S, Balasubramanian S, Guo Y L. 2019. Transposable elements drive rapid phenotypic variation in Capsella rubella. Proceedings of the National Academy of Sciences of the United States of America, 116 (14):6908-6913.
[19]	Sinzelle L, Kapitonov V V, Grzela D P, Jursch T, Jurka J, Izsvák Z, Ivics Z. 2008. Transposition of a reconstructed Harbinger element in human cells and functional homology with two transposon-derived cellular genes. Proceedings of the National Academy of Sciences of the United States of America, 105 (12):4715-4720.
[20]	Stefanov B A, Nowacki M. 2024. The roles of transposable elements in transgenerational inheritance and genome evolution. In:Witzany G ed. Epigenetics in Biological Communication. Cham: Springer Nature Switzerland,369-385.
[21]	Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods. Molecular Biology and Evolution, 28 (10):2731-2739.
[22]	Tang W, Ji Q, Huang Y, Jiang Z, Bao M, Wang H, Lin R. 2013. FAR-RED ELONGATED HYPOCOTYL3 and FAR-RED IMPAIRED RESPONSE1 transcription factors integrate light and abscisic acid signaling in Arabidopsis. Plant Physiology, 163 (2):857-866.
[23]	Wang D, Su Y, Wang X, Lei H, Yu J. 2012. Transposon-derived and satellite-derived repetitive sequences play distinct functional roles in Mammalian intron size expansion. Evolutionary Bioinformatics,8:301-319.
[24]	Wang L, Dou K, Moon S, Tan F J, Zhang Z Z. 2018. Hijacking oogenesis enables massive propagation of LINE and retroviral transposons. Cell, 174 (5):1082. doi: S0092-8674(18)30843-2 pmid: 30057117
[25]	Wang L, Tracy L, Su W, Yang F, Feng Y, Silverman N, Zhang Z Z Z. 2022. Retrotransposon activation during Drosophila metamorphosis conditions adult antiviral responses. Nature Genetics, 54 (12):1933-1945. doi: 10.1038/s41588-022-01214-9 pmid: 36396707
[26]	Xiang Yuanping, Wang Yidan, He Hongjun, Xu Qijiang. 2020. Plant transposable elements and the effects of insertion mutations on flower development in horticultural plants. Acta Horticulturae Sinica, 47 (11):2247-2266. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-0361
	相元萍, 王一丹, 贺洪军, 徐启江. 2020. 植物转座子类型及其插入突变对园艺植物花发育影响的研究进展. 园艺学报, 47 (11):2247-2266.
[27]	Yaakov B, Kashkush K. 2011. Methylation,transcription,and rearrangements of transposable elements in synthetic allopolyploids. Int J Plant Genomics,569826.
[28]	Yang H, Zhang J T, Zhang H W, Cao R X, Tang D L, Wang L J. 2024. 5-Aminolevulinic acid against strawberry Fusarium wilt:Bidirectional regulation of biocontrol agents and pathogens. Horticultural Plant Journal, 10 (6):1349-1361.
[29]	Yao M C, Mao D. 2022. The Harbinger transposon-derived gene PANDA epigenetically coordinates panicle number and grain size in rice. Genes Development, 20 (6):1154-1166.
[30]	Zhang L, Wang R, Xing Y, Xu Y, Xiong D, Wang Y, Yao S. 2021. Separable regulation of POW1 in grain size and leaf angle development in rice. Plant Biotechnol Journal, 19 (12):2517-2531.
[31]	Zhou X, He J, Velanis C N, Zhu Y, He Y, Tang K, Zhu M, Graser L, de Leau E, Wang X, Zhang L, Andy Tao W, Goodrich J, Zhu J, Zhang C. 2021. 3 deacetylation at genes but not TEs in Arabidopsis. Journal of Integrative Plant Biology, 63 (8):1462-1474.

月季Harbinger类转座子基因RcHTD的克隆与功能初探

Cloning and Preliminary Functional Exploration of the Transposon-Derived Gene RcHTD in Rose

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 8

参考文献 31

相关文章 15

编辑推荐

Metrics

本文评价

访问总数:　当日访问总数:　当前在线人数:

[1]	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.
[2]	莫锦夏, 李芳, 熊欣婷, 钟灶发, 彭婷. 枳PtSARD1的特征分析及其抗旱功能初探[J]. 园艺学报, 2025, 52(6): 1399-1411.
[3]	马豫皖, 刘澳, 刘向东, 张雅婧, 董炫克, 李玉帆, 陈己任. 月季‘月月红’RcRAP2.7的克隆及其在非生物胁迫下的表达分析[J]. 园艺学报, 2025, 52(4): 921-932.
[4]	付琪, 王丹, 景维坤, 张颢, 王慧纯, 蹇洪英, 邱显钦, 王其刚, 唐开学, 晏慧君. 月季类胡萝卜素裂解双加氧酶基因RcCCD4在花香合成中的功能[J]. 园艺学报, 2025, 52(3): 623-634.
[5]	阎旭, 李月, 傅小鹏, 宁国贵, 梁梅. 中国古老月季‘月月粉’等位基因不平衡分析与分子标记开发利用[J]. 园艺学报, 2025, 52(2): 365-379.
[6]	任恒泽, 李丹莹, 余亚婷, 吕务云, 郝心愿, 王新超, 王玉春. 植物VIGS载体构建策略研究与应用进展[J]. 园艺学报, 2024, 51(7): 1455-1473.
[7]	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.
[8]	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.
[9]	兰伟, 孟艳琼, 宣云, 朱糠宁, 丁晓浩, 樊德新, 康丽云. 月季新品种‘颍荷’[J]. 园艺学报, 2024, 51(4): 925-926.
[10]	周燕 , 高述民 , 崔荣峰 , 孙丽萍 , 付子豪 , . 月季新品种‘燕京黄’[J]. 园艺学报, 2023, 50(S1): 149-150.
[11]	陆静, 韦素云, 尹佟明, 陈赢男. Type-A细胞分裂素响应调节因子基因家族研究进展[J]. 园艺学报, 2023, 50(9): 1867-1888.
[12]	马梦宇, 胡耀芳, 古琳, 李振坚, 钱永强, 巨关升, 刘俊祥, 孙振元. 瓶插月季枝条光照处理对切花寿命的延长效应[J]. 园艺学报, 2023, 50(6): 1343-1354.
[13]	郭静, 廖满余, 金燕, 马小川, 张芬, 卢晓鹏, 邓子牛, 盛玲. 柑橘转录因子CsbHLH3调控柠檬酸代谢的功能解析[J]. 园艺学报, 2023, 50(5): 947-958.
[14]	刘嘉琦, 公菲菲, 张颢, 景维坤, 瞿素萍, 马男, 高俊平, 孙小明. 月季茉莉酸羧基甲基转移酶基因RhJMT对花瓣衰老的调控[J]. 园艺学报, 2023, 50(5): 1025-1036.
[15]	高俊平, 马男, 周厚高, 王嘉曦, 张常青, 李永红, 周晓锋, 孙小明. 切花月季新品种‘菲韵’[J]. 园艺学报, 2023, 50(4): 915-916.

月季Harbinger类转座子基因RcHTD的克隆与功能初探

Cloning and Preliminary Functional Exploration of the Transposon-Derived Gene RcHTD in Rose

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 8

参考文献 31

相关文章 15

编辑推荐

Metrics

本文评价

访问总数: 当日访问总数: 当前在线人数:

访问总数:　当日访问总数:　当前在线人数: