Cloning and Functional Analysis of RcMADS2 Gene in Rosa chinensis 'Viridiflora'
ZHANG Jin-Feng1, ZHANG Hua2, SONG Li-Na2, ZHANG Hua-Li2, LIAO Hui3, YANG Yi-Peng2, JI Peng1,*
1 College of Gardening, Heilongjiang Bayi Agricultural University, Daqing 163319, China; 2 Beijing Academy of Forestry and Landscape Architecture, Beijing 100102, China; 3 College of Landscape Architecture, Beijing University of Agriculture, Beijing 102206, China
Abstract：The MADS-box gene family can determine floral morphogenesis and play an important role in floral organ development. Based on the previous transcriptome analysis of Rosa chinensis 'Viridiflora', the MADS-box transcription factor-RcMADS2 related to floral organ variation was screened. In order to explore the biological function of this gene, this study cloned the RcMADS2, analyzed its biological information and expression pattern, and further verified its function by subcellular localization, heterologous overexpression and virus-induced gene silencing. The results showed that the CDS sequence of RcMADS2 was 762 bp, encoding 253 amino acids. It was predicted that the protein was an alkaline and unstable hydrophilic protein with highly conserved MADS-MEF2-like and K-box domains. The protein sequence had the highest similarity with the homologous sequence of R. rugosa. Phylogenetic tree analysis showed that RcMADS2 and E-class genes of Arabidopsis thaliana MADS-box family were divided into the same branch, and the genetic relationship was similar. By analyzing the expression pattern, it was found that the RcMADS2 gene had the highest expression level in the sepals of R. chinensis 'Viridiflora', while it was basically not expressed in the leaves. Subcellular localization results showed that RcMADS2 was located in the nucleus, which was consistent with the prediction. Overexpression of RcMADS2 in A. thaliana showed that the number of sepals in A. thaliana positive plants changed and the growth potential of the plants decreased, and the expression of RcMADS2 in the plants increased significantly (P<0.05). The qRT-PCR results showed that the expression of RcMADS2 in the silenced plants decreased, but no obvious phenotypic changes were found. This study preliminarily proved that the RcMADS2 gene of R. chinensis 'Viridiflora' was an E-class gene, which was involved in the regulation of floral organ development, and provides relevant theories for the study of rose flower development in the later stage.
[1] 董佳乐. 2018. 草莓FaMADS1和FaMADS2基因的克隆、鉴定和功能初探[D]. 硕士毕业论文, 安徽农业大学, 导师: 方从兵, pp. 21-26. (Dong J L.2018. Cloning, identification and functional analysis of FaMADS1 and FaMADS2 genes in Fragaria × ananassa[D]. Thesis for M.S., Anhui Agricultural University, Supervisor: Fang C B, pp. 21-26.) [2] 黄善武, 葛红, 陈琰芳. 1990. 绿色花月季的研究[J]. 中国园林,(02): 47-48+59. (Huang S W, Ge H, Chen Y F.1990. Study on green rose[J].Chinese Gardens,(02): 47-48+59.) [3] 韩迎春. 2022. 小麦转录因子TaMADS2参与磷胁迫响应的机制及其功能研究[D]. 博士毕业论文, 河南农业大学, 导师: 谭金芳, 郑文明, pp. 40-41. (Han Y C.2022. The mechanism and function of wheat transcription factor TaMADS2 involved in phosphorus stress response[D].Thesis for Ph. D., Henan Agricultural University, Supervisor: Tan J F, Zheng W M, pp. 40-41.) [4] 赖恭梯, 潘若, 贺丽媛, 等. 2024. 山茶花SEPALLATA (SEP)家族基因克隆及表达分析[J]. 福建农业科技, 55(12): 28-36. (Lai G D, Pan R, He L Y, et al.2024. Cloning and expression analysis of SEPALLATA (SEP) family genes in Camellia japonica[J]. Fujian Agricultural Science and Technology, 55(12): 28-36.) [5] 李丹丹. 2017. 石竹花发育E类基因的克隆及功能分析[D]. 硕士毕业论文, 华中农业大学, 导师: 傅小鹏. pp. 41-47. (Li D D.2017. Cloning and functional analysis of class E genes in carnation flower development[D]. Thesis for M.S., Huazhong Agricultural University, Supervisor: Fu X P, pp. 41-47.) [6] 李文辰, 刘鑫, 康越, 等. 2023. TRV病毒诱导大豆基因沉默体系优化及应用[J]. 生物技术通报, 39(07): 143-150. (Li W C, Liu X, Kang Y, et al.2023. Optimization and application of Tobacco rattle virus-induced gene silencing system in soybean[J]. Biotechnology Bulletin, 39(07): 143-150.) [7] 李玉帆, 杜姣林, 张翔, 等. 2024. 月季'月月红' RcTPS1a基因在调控植物开花时间中的应用. 湖南省, CN118745435A[P]. (Li Y F, Du J L, Zhang X, et al.2024. Application of RcTPS1a gene in regulating flowering time of Rosa chinensis Jacq. Hunan Province, CN118745435A[P].) [8] 李卓姣. 2021. 大花紫薇花发育相关4个MADS-box基因功能研究[D]. 硕士毕业论文, 北京林业大学, 导师: 张启翔. pp. 61-62. (Li Z J.2021. Functional study of four MADS-box genes related to flower development of Lagerstroemia indica[D]. Thesis for M.S., Beijing Forestry University, Supervisor: Zhang Q X, pp. 61-62.) [9] 彭洁, 刘引, 武荣花, 等. 2023. 重瓣花及其分子机制的研究进展[J]. 中国农学通报, 39(19): 65-72. (Peng J, Liu Y, Wu R H, et al.2023. Research progress on double flower and its molecular mechanism[J]. China Agricultural Bulletin, 39(19): 65-72.) [10] 王天野, 杨梦情, 马慧, 等. 2025. 滇水金凤花距发育相关基因MADS-EJ2和MADS2的克隆及表达分析[J]. 山东农业科学, 1-10. (Wang T Y, Yang M Q, Ma H, et al.2025. Cloning and expression analysis of MADS-EJ2 and MADS2 genes related to floral development in Impatiens uliginosa[J]. Shandong Agricultural Sciences, 1-10.) [11] 王凯璇. 2023. '卡罗拉'月季花器官发育相关基因RhSEP3的克隆与功能探究[D]. 硕士毕业论文, 河南农业大学, 导师: 石力匀; 张晖. pp. 39-40. (Wang K X.2023. Cloning and functional analysis of RhSEP3 gene related to floral organ development in Rosa 'Carola'[D]. Thesis for M.S., Henan Agricultural University, Supervisor: Shi L J, Zhang H, pp. 39-40.) [12] 王莹, 穆艳霞, 王锦. 2021. MADS-box基因家族调控植物花器官发育研究进展[J]. 浙江农业学报, 33(06): 1149-1158. (Wang Y, Mu Y X, Wang J.2021. Research progress of MADS-box gene family regulating plant floral organ development[J]. Zhejiang Agricultural Journal, 33(06): 1149-1158.) [13] 吴彦庆, 汤寓涵, 赵大球, 等. 2017. 芍药内外花瓣发育形成相关基因片段的生物信息学及组织表达分析[J]. 中国农业大学学报, 22(12): 53-63. (Wu Y Q, Tang Y H, Zhao D Q, et al.2017. Bioinformatics and tissue expression analysis of genes affecting the formation and development of inner/outer petal in Paeonia lactiflora[J]. Journal of China Agricultural University, 22(12): 53-63.) [14] 杨祥燕, 蔡元保, 张治礼, 等. 2016. 菠萝MADS-box基因AcMADS2的克隆与表达分析[J]. 植物生理学报, 52(09): 1406-1412. (Yang X Y, Cai Y B, Zhang Z L, et al.2016. Cloning and expression analysis of MADS-box gene AcMADS2 in Ananas comosus (L.)[J]. Acta Phytophysiology Sinica, 52(09): 1406-1412.) [15] 杨晓颖, 刘菊华, 徐碧玉, 等. 2008. 香蕉MuMADS2的克隆、序列分析和表达分析[J]. 分子植物育种, (04): 781-786. (Yang X Y, Liu J H, Xu B Y, et al. 2008. Cloning, sequence and expression analysis of banana MuMADS2[J]. Molecular Plant Breeding, (04): 781-786.) [16] 张景霞, 王芙蓉, 高阳, 等. 2015. VIGS技术及其在棉花功能基因组研究中的应用进展[J]. 棉花学报, 27(05): 469-473. (Zhang J X, Wang F R, Gao Y, et al.2015. VIGS technology and its application in cotton functional genomics research[J]. Cotton Journal, 27(05): 469-473.) [17] Adal A M, Binson E, Remedios L, et al.2021. Expression of lavender AGAMOUS-like and SEPALLATA3-like genes promote early flowering and alter leaf morphology in Arabidopsis thaliana[J].Planta, 254(3): 54-54. [18] Bendahmane M, Dubois A, Raymond O, et al.2013. Genetics and genomics of flower initiation and development in roses[J]. Journal of Experimental Biology, 64(4): 847-857. [19] Bowman J L, Smyth D R, Meyerowitz E M.1991. Genetic interactions among floral homeotic genes of Arabidopsis[J]. Development, 112(1): 1-20. [20] Ditta G, Pinyopich A, Robles P, et al.2004. The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity[J]. Current Biology, 14(21): 1935-1940. [21] Jing D L, Chen W W, Shi M, et al.2020. Ectopic expression of an Eriobotrya japonica APETALA3 ortholog rescues the petal and stamen identities in Arabidopsis ap3-3 mutant[J]. Biochemical and Biophysical Research Communications, 523(1): 33-38. [22] Liu J Y, Fu X D, Dong Y W, et al.2018. MIKC supC/sup-type MADS-box genes in Rosa chinensis: The remarkable expansion of ABCDE model genes and their roles in floral organogenesis[J]. Horticulture Research, 5(1): 25. [23] Martino G, Pan I, Emmanuel E, et al.2006. Functional analyses of two tomato APETALA3 genes demonstrate diversification in their roles in regulating floral development[J].Plant Cell, 18(8): 1833-1845. [24] Soza V L, Snelson C D, Hewett Hazelton K D, et al.2016. Partial redundancy and functional specialization of E-class SEPALLATA genes in an early-diverging eudicot[J]. Developmental Biology, 419(1): 143-155. [25] Yan H, Zhang H, Wang Q, et al.2016. The Rosa chinensis cv. 'Viridiflora' phyllody phenotype is associated with misexpression of flower organ identity genes[J]. Front in Plant Science, 7: 996. [26] Zhang J F, Liao H, Yang Y P, et al.2025. The function of RcAG2 and RcFUL in the flower shape change of Rosa chinensis 'Viridiflora'[J]. Plants (Basel), 15(1): 11.
