‘凤丹’牡丹PoFD基因克隆及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0004

摘要/Abstract

摘要：

Flowering Locus D（FD）基因属于bZIP转录因子家族，与FT基因相互作用，在促进植物开花等方面发挥重要作用。本研究以‘凤丹’牡丹为材料，基于‘凤丹’三代全长转录组测序结果，采用RT-qPCR技术克隆PoFD基因，并进行生物信息学和表达模式分析。结果表明，克隆到的PoFD基因含有一个2 712 bp完整的ORF框，编码903个氨基酸。PoFD蛋白分子式为C₈₂₆₁H₁₃₇₂₂N₂₇₁₂O₃₄₆₈S₅₅₂，理论等电点（pI）为4.88，为亲水蛋白，无跨膜结构，二级结构中无规则卷曲和α-螺旋所占比例较高，β-转角仅占少部分。荧光定量分析发现，PoFD基因在叶片中表达量最高，推测PoFD基因可能主要作用于叶片调控牡丹花期。牡丹不同花发育时期，PoFD基因在半开期的表达量较高，推测PoFD基因在‘凤丹’开花的中期发挥功能。不同浓度油菜素内酯（brassinosteroids，BR）激素喷施处理下，‘凤丹’牡丹花期具有不同程度的延迟，并且PoFD基因的表达均有所下降，表明PoFD基因可能响应BR调控牡丹花期。本研究为进一步研究PoFD基因在牡丹花期调控中的作用提供理论参考。

关键词: ‘凤丹’牡丹, 花期调控, PoFD, 基因克隆, 生物信息学分析, 表达分析

Abstract:

Flowering Locus D（FD）gene, a member of the bZIP transcription factor family, plays an important role in promoting plant flowering interacting with the FT gene via interaction with FT gene. In this study, tree peony（Paeonia ostii）variety ‘Fengdan’ was used as the material, the PoFD gene was cloned by RT-qPCR technology based on the results of the three-generation full-length transcriptome sequencing of ‘Fengdan’, and the bioinformatics and expression patterns analysis were performed on it. The results showed that the cloned PoFD gene contained a 2 712 bp complete ORF frame, encoding 903 amino acids. The molecular formula of PoFD protein is C₈₂₆₁H₁₃₇₂₂N₂₇₁₂O₃₄₆₈S₅₅₂, and the theoretical isoelectric point（pI）is 4.88. It is a hydrophilic protein and has no transmembrane structure. There are a high proportion of random coils and α-helices in secondary structure, while β-turns account for only a small part. RT-qPCR analysis demonstrated the expression of PoFD gene was the highest in leaves, thus speculating that PoFD gene may mainly function in leaves to regulate the flowering stage of tree peony. In different flower development stages, the expression of PoFD was the highest at the half-open stage, inferring that the PoFD gene may play the role at the middle flowering stage. Compared with the control group, the flowering stage of ‘Fengdan’ delayed at varying degrees while treated with varied concentrations of brassinosteroid（BR）, and the expression of PoFD gene decreased, indicating that PoFD gene may regulate tree peony flowering period in response to BR. This study provides a theoretical reference for the follow-up study of the role of PoFD gene in the regulation of tree peony flowering.

Key words: Paeonia ostii ‘Fengdan’, flowering regulating, PoFD, gene cloning, bioinformatics analysis, expression analysis

张琳, 魏祯祯, 宋程威, 郭丽丽, 郭琪, 侯小改, 王华芳. ‘凤丹’牡丹PoFD基因克隆及表达分析[J]. 生物技术通报, 2022, 38(11): 104-111.

ZHANG Lin, WEI Zhen-zhen, SONG Cheng-wei, GUO Li-li, GUO Qi, HOU Xiao-gai, WANG Hua-fang. Cloning and Expression Analysis of PoFD Gene from Paeonia ostii ‘Fengdan’[J]. Biotechnology Bulletin, 2022, 38(11): 104-111.

图/表 9

表1 引物序列

Table 1 Primer sequence

引物名称 Primer name	序列 Sequence（5'-3'）	扩增目的 Amplification purpose
PoFD-F	TTTACCGGACCTGAACAC	克隆PoFD基因 Cloning of PoFD gene
PoFD-R	ACCCAACACCCTCATACA	克隆PoFD基因 Cloning of PoFD gene
PoFD-RT-F	ATTGTAGGCGGTAGGAGCAC	PoFD基因RT-qPCR分析 RT-qPCR assay of PoFD gene
PoFD-RT-R	TATCAAGCACGGTCGCTGT	PoFD基因RT-qPCR分析 RT-qPCR assay of PoFD gene
Actin-F Actin-R	GGTCTATTCTTGCTTCCCTCAG GAACTCACTATCAAACCCTCCAG	RT-qPCR内参基因 RT-qPCR reference gene

图1 ‘凤丹’PoFD基因凝胶电泳图 M：DL5000 DNA maker；1：PoFD基因的扩增产物

Fig. 1 Electrophoresis diagram of ‘Fengdan’ PoFD gene M：DL5000 DNA marker. 1：Amplified product of PoFD gene

图2 ‘凤丹’PoFD蛋白磷酸化位点分析

Fig. 2 Analysis of phosphorylation sites in ‘Fengdan’ PoFD protein

图3 PoFD蛋白二级结构预测

Fig. 3 Secondary structure prediction of PoFD protein

图4 PoFD蛋白三级结构预测

Fig. 4 Tertiary structure prediction of PoFD protein

图5 ‘凤丹’PoFD蛋白系统进化树分析 Juglans regia：胡桃，XP 018851616.1；Carya illinoinensis：美国山核桃，KAG2723443.1；Morella rubra：杨梅，KAB1219032.1；Carpius fangiana：川黔千金榆，KAE799151.1；Csatanea mollissima：板栗，KAF3951949.1；Quercus lobata：阔叶栎，XP 303929184.1；Quercus suber：栓皮栎，XP 023912111.1；Prunus dulcis：扁桃，XP 034205433.1；Ziziphus jujuba：酸枣，XP 015866666.1；Vitis riparia：葡萄，XP 034703729.1

Fig. 5 Phylogenetic tree analysis of ‘Fengdan’ PoFD protein

图6 PoFD基因在‘凤丹’不同组织中的表达分析不同小写字母表示在0.05水平差异显著，下同

Fig. 6 Expression analysis of PoFD gene in different tissues of ‘Fengdan’ Different lowercases indicate significant differences at the 0.05 level. The same below

图7 PoFD基因在‘凤丹’不同花发育时期的表达分析

Fig. 7 Analysis of expression of PoFD gene in different flower development stages of ‘Fengdan’

图8 PoFD基因在不同浓度BR激素处理下的表达分析

Fig. 8 Expression analysis of PoFD gene under different concentrations of BR hormone treatment

参考文献 32

[1]	洪德元, 潘开玉. 芍药属牡丹组的分类历史和分类处理[J]. 植物分类学报, 1999, 37(4):351-368.
	Hong DY, Pan KY. Taxonomical history and revision of Paeonia sect. Moutan(Paeoniaceae)[J]. Acta Phytotaxon Sin, 1999, 37(4):351-368.
[2]	Zhang JJ, Shu QY, Liu ZA, et al. Two EST-derived marker systems for cultivar identification in tree peony[J]. Plant Cell Rep, 2012, 31(2):299-310. doi: 10.1007/s00299-011-1164-1 pmid: 21987120
[3]	杨礼通, 张文静, 雷映雪, 等. 推迟或提前牡丹花期的调控研究进展[J]. 四川农业科技, 2021(8):80-81, 85.
	Yang LT, Zhang WJ, Lei YX, et al. Research progress on the regulation of delaying or advancing the flowering period of tree peony[J]. Sichuan Agric Sci Technol, 2021(8):80-81, 85.
[4]	石丰瑞. 牡丹长日照途径关键基因PsFT以及其上、下游基因PsCO、PsSOC1的克隆与表达分析[D]. 北京: 中国农业科学院, 2013.
	Shi FR. Cloning and expression analysis of key gene PsFT of long-day photoperiod pathway and its upstream and downstream genes PsCO, PsSOCl in tree peony(Paeonia suffruticosa andr. )[D]. Beijing: Chinese Academy of Agricultural Sciences, 2013.
[5]	康圣楠, 陈曦, 张雪敏, 等. 百合花期调控研究进展[J/OL]. 分子植物育种, 2021. https://kns.cnki.net/kcms/detail/46.1068.S.20210930.1435.008.html.
	Kang SN, Chen X, Zhang XM, et al. Research progress on regulation of flowering stage of Lilium brownii var[J/OL]. Mol Plant Breed, 2021. https://kns.cnki.net/kcms/detail/46.1068.S.20210930.1435.008.html.
[6]	李瑞英, 任崇勇, 张婷. 影响牡丹花期的主要气象因素及预测方法[J]. 现代农业科技, 2016(13):244-245.
	Li RY, Ren CY, Zhang T. Influencing meteorological factors on peony flowering and prediction method[J]. Mod Agric Sci Technol, 2016(13):244-245.
[7]	周华. 基于转录组比较的牡丹开花时间基因发掘[D]. 北京: 北京林业大学, 2015.
	Zhou H. Discovery of genes associated with flowering time in tree peonies based on transcriptome comparison[D]. Beijing: Beijing Forestry University, 2015.
[8]	张彦楠, 于秀立, 吕新华, 等. 棉花bZIP转录因子FD基因的发掘和GhFD基因的组织表达分析[J]. 分子植物育种, 2016, 14(9):2250-2260.
	Zhang YN, Yu XL, Lv XH, et al. Genome-wide identification of bZIP transcription factor FD genes and expression patterns analysis of GhFD genes in cotton[J]. Mol Plant Breed, 2016, 14(9):2250-2260.
[9]	Varkonyi-Gasic E, Moss SMA, Voogd C, et al. Homologs of FT, CEN and FD respond to developmental and environmental signals affecting growth and flowering in the perennial vine kiwifruit[J]. New Phytol, 2013, 198(3):732-746. doi: 10.1111/nph.12162 pmid: 23577598
[10]	Ryu JY, Lee HJ, Seo PJ, et al. The Arabidopsis floral repressor BFT delays flowering by competing with FT for FD binding under high salinity[J]. Mol Plant, 2014, 7(2):377-387. doi: 10.1093/mp/sst114 URL
[11]	Kawamoto N, Sasabe M, Endo M, et al. Calcium-dependent protein kinases responsible for the phosphorylation of a bZIP transcription factor FD crucial for the florigen complex formation[J]. Sci Rep, 2015, 5:8341. doi: 10.1038/srep08341 pmid: 25661797
[12]	蔡芳芳. 二球悬铃木FT和FD同源基因的功能分析及选择性剪切初探[D]. 武汉: 华中农业大学, 2019.
	Cai FF. Functional and alternative splicing analysis of FT and FD homologous genes in Platanus acerifolia[D]. Wuhan: Huazhong Agricultural University, 2019.
[13]	曹青霞. 茶树CsFD基因克隆及其与CsFT基因在成花转变中的功能研究[D]. 西安: 陕西师范大学, 2018.
	Cao QX. The CsFD gene cloning of tea plant and the study of its function with CsFT gene in flowering transition[D]. Xi’an: Shaanxi Normal University, 2018.
[14]	袁敏, 葛伟娜, 王莉, 等. 拟南芥蛋白激酶SnRK1. 1对转录因子FD的磷酸化分析[J]. 华北农学报, 2017, 32(4):37-41. doi: 10.7668/hbnxb.2017.04.006
	Yuan M, Ge WN, Wang L, et al. Protein kinase SnRK1. 1 phosphorylates transcriptional factor FD in Arabidopsis thaliana[J]. Acta Agric Boreali Sin, 2017, 32(4):37-41.
[15]	Taoka KI, Ohki I, Tsuji H, et al. 14-3-3 proteins act as intracellular receptors for rice Hd3a florigen[J]. Nature, 2011, 476(7360):332-335. doi: 10.1038/nature10272 URL
[16]	Li ZC, Ou Y, Zhang ZC, et al. Brassinosteroid signaling recruits histone 3 lysine-27 demethylation activity to FLOWERING LOCUS C chromatin to inhibit the floral transition in Arabidopsis[J]. Mol Plant, 2018, 11(9):1135-1146. doi: 10.1016/j.molp.2018.06.007 URL
[17]	侯倩倩. MdFD基因的功能验证及苹果早花相关基因的表达分析[D]. 北京: 中国农业科学院, 2018.
	Hou QQ. Functional verification of MdFD gene and expression analysis of early flower related genes in apple[D]. Beijing: Chinese Academy of Agricultural Sciences, 2018.
[18]	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4):402-408. doi: 10.1006/meth.2001.1262 pmid: 11846609
[19]	Blümel M, Dally N, Jung C. Flowering time regulation in crops—what did we learn from Arabidopsis?[J]. Curr Opin Biotechnol, 2015, 32:121-129. doi: 10.1016/j.copbio.2014.11.023 URL
[20]	Vollrath P, Chawla HS, Schiessl SV, et al. A novel deletion in FLOWERING LOCUS T modulates flowering time in winter oilseed rape[J]. Theor Appl Genet, 2021, 134(4):1217-1231. doi: 10.1007/s00122-021-03768-4 pmid: 33471161
[21]	Alves MS, Dadalto SP, Gonçalves AB, et al. Plant bZIP transcription factors responsive to pathogens:a review[J]. Int J Mol Sci, 2013, 14(4):7815-7828. doi: 10.3390/ijms14047815 URL
[22]	Lee BJ, Park CJ, Kim SK, et al. In vivo binding of hot pepper bZIP transcription factor CabZIP1 to the G-box region of pathogenesis-related protein 1 promoter[J]. Biochem Biophys Res Commun, 2006, 344(1):55-62. doi: 10.1016/j.bbrc.2006.03.153 URL
[23]	段亚宾. 牡丹开花相关基因的克隆和表达分析[D]. 洛阳: 河南科技大学, 2015.
	Duan YB. Cloning and expression of flowering genes in tree peony[D]. Luoyang: Henan University of Science and Technology, 2015.
[24]	罗轩, 丛汉卿, 李丽, 等. 蜻蜓凤梨FLD同源基因的克隆及表达分析[J]. 分子植物育种, 2013, 11(3):371-378.
	Luo X, Cong HQ, Li L, et al. Cloning and expression analysis of FLD homologous gene from Aechmea fasciata[J]. Mol Plant Breed, 2013, 11(3):371-378.
[25]	罗玉秀, 张生萍, 许唱唱, 等. 特早熟春性甘蓝型油菜sBnFLD基因的克隆及表达[J]. 西北农林科技大学学报:自然科学版, 2016, 44(11):90-96.
	Luo YX, Zhang SP, Xu CC, et al. Cloning and expression of sBnFLD gene from spring rapeseed(Brassica napus L.)[J]. J Northwest A&F Univ Nat Sci Ed, 2016, 44(11):90-96.
[26]	Tsuji H, Nakamura H, Taoka KI, et al. Functional diversification of FD transcription factors in rice, components of florigen activation complexes[J]. Plant Cell Physiol, 2013, 54(3):385-397. doi: 10.1093/pcp/pct005 pmid: 23324168
[27]	Wigge PA, Kim MC, Jaeger KE, et al. Integration of spatial and temporal information during floral induction in Arabidopsis[J]. Science, 2005, 309(5737):1056-1059. doi: 10.1126/science.1114358 URL
[28]	Li CX, Dubcovsky J. Wheat FT protein regulates VRN1 transcription through interactions with FDL2[J]. Plant J, 2008, 55(4):543-554. doi: 10.1111/j.1365-313X.2008.03526.x URL
[29]	Andrés F, Coupland G. The genetic basis of flowering responses to seasonal cues[J]. Nat Rev Genet, 2012, 13(9):627-639. doi: 10.1038/nrg3291 pmid: 22898651
[30]	Dutta S, Deb A, Biswas P, et al. Identification and functional characterization of two bamboo FD gene homologs having contrasting effects on shoot growth and flowering[J]. Sci Rep, 2021, 11(1):7849. doi: 10.1038/s41598-021-87491-6 URL
[31]	Jang S, Choi SC, Li HY, et al. Functional characterization of Phalaenopsis aphrodite flowering genes PaFT1 and PaFD[J]. PLoS One, 2015, 10(8):e0134987.
[32]	张玲. 枇杷花期调控的分子生物学研究[D]. 广州: 华南农业大学, 2016.
	Zhang L. Studies on molecular mechanism of loquat flowering time regulation[D]. Guangzhou: South China Agricultural University, 2016.