中国水仙<i>FT</i>基因的表达及功能分析

李婷婷; 潘秋宇; 吴菁华

doi:10.19303/j.issn.1008-0384.2023.12.005

中国水仙FT基因的表达及功能分析

doi: 10.19303/j.issn.1008-0384.2023.12.005

1.
福建农林大学园艺学院，福建　福州　350002

基金项目: 福建省自然科学基金项目（2021J01087）；福建省科技计划星火项目（2021S0068）

详细信息

作者简介:
李婷婷（1994 —），女，硕士，主要从事园艺植物遗传育种相关研究，E-mail：15236174388@qq.com

通讯作者:
吴菁华（1978 —），女，博士，副教授，主要从事园艺植物遗传育种相关研究，E-mail：wjhham@163.com

中图分类号: S682.21
计量
- 文章访问数: 326
- HTML全文浏览量: 159
- PDF下载量: 52
- 被引次数: 0
出版历程
- 收稿日期: 2023-09-12
- 修回日期: 2023-11-02
- 网络出版日期: 2024-01-06
- 刊出日期: 2023-12-28

Expressions and Functions of Flowering Locus Ts in Narcissus tazetta var. chinensis Roem

1.
College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China

摘要

摘要: 目的开花基因座T（Flowering Locus T，FT）基因广泛参与植物的生长发育，在调控开花、地下茎发育、种子萌发和逆境胁迫等过程中发挥重要作用。揭示FT基因在中国水仙（Narcissus tazetta var. chinensis Roem）中的表达模式和功能可为中国水仙花期调控提供理论依据。方法基于中国水仙转录组数据，对FT 基因进行筛选，获得4个中国水仙FT基因。利用荧光定量PCR技术分析它们在中国水仙不同组织和花芽分化不同时期的表达模式，并在拟南芥（Arabidopsis thaliana）中超量表达。利用荧光定量PCR技术分析转NtFT1和NtFT4基因拟南芥中SUPPRESSOR OF OVEREXPRESSION OF CO 1（SOC1）、LEAFY（LFY）和APETALA 1（AP1）基因表达水平。结果克隆了4个中国水仙FT同源基因NtFT1、NtFT2、NtFT3和NtFT4，除NtFT3外，中国水仙NtFT都具有FT的保守基序；系统进化分析显示，NtFT1属于FT-like I进化枝，NtFT2、NtFT3和NtFT4同属于FT-like II类进化枝。不同的NtFT基因在中国水仙组织器官和花芽分化不同时期的表达模式存在差异：NtFT1和NtFT3在花中表达量最高，NtFT2在叶片中表达量最高，NtFT4在鳞片中的表达量最高；NtFT1在花芽分化过程中均呈先上升后下降的趋势，NtFT2整个主芽分化过程中变化幅度不大，NtFT3和NtFT4在整个花芽分化期表达量都较低。异位转化拟南芥结果显示，与野生型拟南芥相比，过表达NtFT1和NtFT2的拟南芥提早开花，过表达NtFT3拟南芥开花时间与野生型植株无明显差异，转NtFT4基因的拟南芥植株推迟开花。过表达NtFT1拟南芥中SOC1、LFY和AP1基因表达量上升。结论在中国水仙中存在多个FT基因，在调控开花的功能上存在差异，NtFT1促进开花，NtFT4抑制开花。
- 中国水仙 /
- FT同源基因 /
- 花期调控 /
- 异位表达
Abstract: Objective Expressions and functions of Flowering Locus T (FT), the genes widely involved in plant growth, flowering regulation, root development, and seed germination, in Chinese narcissus were studied. Method Four FTs were identified from the transcriptome data on Narcissus tazetta var. chinensis Roem by bioinformatics analysis. Expressions of the genes in various tissues and flower buds at differentiation stages were determined by fluorescence quantitative PCR and further confirmed by overexpressing it in Arabidopsis thaliana. Result The 4 homologous FTs, i.e., NtFT1, NtFT2, NtFT3, and NtFT4, were cloned using RT-PCR. All of them, except NtFT3, had conserved motifs. Phylogenetically; NtFT1 belonged to the FT-like I branch, while NtFT2, NtFT3, and NtFT4, to the FT-like II branch. In tissues, organs, and flower buds at differentiation stages, they expressed differently. The highest expressions of NtFT1 and NtFT3 were in the flowers, those of NtFT2 in the leaves, and NtFT4 in the scales. At different flower bud differentiation stages, the expressions differed significantly as well, as NtFT1 decreased followed as an increase during differentiation, but NtFT2 changed little throughout. The expressions of NtFT3 and NtFT4 were relatively low in the entire flower bud differentiation period. The ectopic transformation of A. thaliana showed the overexpressed NtFT1 and NtFT2 led to early flowering in comparison to the wild type. But no effect was associated with NtFT3, but delayed flowering was found on the transgenic NtFT4 Arabidopsis plants. In addition, the expressions of SOC1, LFY, and AP1 in the Arabidopsis plants rose with NtFT1 overexpression. Conclusion There were multiple FTs in Chinese narcissus that differed in regulating the flowering—NtFT1 promoted, while NtFT4 inhibited, the process.
- Narcissus tazetta var. chinensis Roem /
- homologous FTs /
- flowering regulation /
- ectopic expression

HTML全文

图 1 NtFT与其他物种FT氨基酸序列的多重比较

Figure 1. Multiple comparison on amino acid sequences of NtFT and FTs of other species

下载: 全尺寸图片幻灯片

图 2 NtFT与其他植物FT 蛋白的系统进化树分析

Figure 2. Phylogenetic trees of NtFT and FTs of other species

下载: 全尺寸图片幻灯片

图 3 NtFT基因在不同组织中的qRT-PCR表达分析

不同小写字母表示同一组织部位不同基因间差异显著（P＜0. 05）。

Figure 3. qRT-PCR on NtFTs in different tissues

Datas with different lowercase letters indicate significant differences between different gene in same tissue (P＜0.05).

下载: 全尺寸图片幻灯片

图 4 NtFT在中国水仙花芽不同分化阶段的表达分析

不同小写字母表示同一发育时间不同基因差异显著（P＜0. 05）。

Figure 4. qRT-PCR on NtFTs from N. tazetta during flower buds at differentiation stages

Datas with different lowercase letters indicate significant differences between genes at same development time (P＜0.05).

下载: 全尺寸图片幻灯片

图 5 NtFT基因在拟南芥的异位表达

A：过表达NtFT基因拟南芥开花情况；B：过表达 NtFT 基因拟南芥开花性状数据。*、**表示与WT相比差异显著（P＜0.05）或极显著（P＜0.01）。图6同。

Figure 5. Ectopic expressions of NtFTs in A. thaliana

A: Flowering of A. thaliana with overexpressed NtFT; B: Flowering traits data of A. thaliana with overexpressed NtFT; * and ** indicate significant difference(P＜0.05) and (P＜0.01), respectively. Same for Fig. 6.

下载: 全尺寸图片幻灯片

图 6 转基因拟南芥中AtLFY、AtSOC1和AtAP1的表达分析

Figure 6. Expressions of AtLFY, AtSOC1, and AtAP1 in transgenic plants

下载: 全尺寸图片幻灯片

表 1 克隆中国水仙NtFT基因引物

Table 1. Primers for cloning NtFT of N. tazetta

引物名称 Primer name	上游引物序列（5′-3′） Upstream primer sequence (5′-3′)	下游引物序列（5′-3′） Downstream primer sequence (5′-3′)
NtFT1	TTTCCGCTTATATCTCTTCTGGGAC	TCGGGAAGTAGCAAGACGATCAAAC
NtFT2	ATGTTGAGAGAGAGGGTACC	TCAGCAAAGTCCTGAGAACCTTCTT
NtFT3	GAAGTAGTCATGTTGAGAGAGAGG	GTATCACATATTGCATGGCTTAGG
NtFT4	GGTTAAGAGACAGAATGCCGAT	TTTATGTCATTTATCGTCTG CTAG

下载: 导出CSV

表 2 FT基因表达qRT-PCR引物

Table 2. Primers for qRT-PCR of FT gene

引物名称 Primer name	上游引物序列（5’-3’） Upstream primer sequence (5′-3′)	上游引物序列（5’-3’） Downstream primer sequence (5′-3′)
NtActin	GTTGACCCACCACTAAGAACAATG	TGCCCAGAAGTGCTATTCCAG
QNtFT1	CCAGCCAAAGGTTGAAGTCG	CCCTGTGGTTCCTGGTATG
QNtFT2	CTTATGAGAGCCCTCGAACACC	CGCACACAGTTTGTTGAACTTCC
QNtFT3	CGCACACAGTTTGTTGAACTTCC	CACTGGTTGGTGACAGACATACC
QNtFT4	TGGCAGGATGCGATGCAAGA	CACGATGCGATGAATCCCCGA

下载: 导出CSV

表 3 pSAK277表达载体构建引物

Table 3. Primers for constructing pSAK277 expression vector

引物名称 Primer name	上游引物序列（5′-3′） Upstream primer sequence (5′-3′)	下游引物序列（5′-3′） Upstream primer sequence (5′-3′)
277NtFT1	ACTAGTGGATCCAAAGAATTCATGA GTAGGGATCCTTTGGTTATTG	AGAAGTACTCTCGAGAAGCTT TTAGGGGTACATCCTCCGGCCACCA
277NtFT2	ACTAGTGGATCCAAAGAATTCAGTTGA GAGAGAGGGTACCAAGGG	AGAAGTACTCTCGAGAAGCTTT CAGCAAAGTCCTGAGAACCTTCTT
277NtFT3	ACTAGTGGATCCAAAGAATTC ATGTTGAGAG AGAGGGTACC	AGAAGTACTCTCGAGAAGCTT TCACATATTG CATGGCTTAG
277NtFT4	ACTAGTGGATCCAAAGAATTCAT GCCGATACTGGGACAAGT	AGAAGTACTCTCGAGAAGCTTTC AGAACCTTCTTCCTCCGCA

下载: 导出CSV

表 4 转基因拟南芥的不同基因qRT-PCR引物

Table 4. Primers for qRT-PCR of different genes in transgenic A. thaliana

引物名称 Primer name	上游引物序列（5’-3’） Upstream primer sequence (5′-3′)	上游引物序列（5’-3’） Upstream primer sequence (5′-3′)
Actin	GCTGAGAGTTGATGGTGTGCT	GGATACCCTTTCGCAGATAGAG
AtLFY	GTTAGGTTTTACGGCGAGCA	GCAATCGTCTCCGTTCAGC
AtAP1	ACCAAATCCAGCATCCTTACA	TCAAGAGTCAGTTCGAGATCATTC
AtSOC1	CTCTCAGTGCTTTGTGATGCT	CGATTGAGCATGTTCCTATGCC

下载: 导出CSV

参考文献(36)

[1]	FORNARA F, DE MONTAIGU A, COUPLAND G. SnapShot: Control of flowering in Arabidopsis [J]. Cell, 2010, 141(3): 550−550.e2. doi: 10.1016/j.cell.2010.04.024
[2]	张艺能, 周玉萍, 陈琼华, 等. 拟南芥开花时间调控的分子基础 [J]. 植物学报, 2014, 49(4):469−482. doi: 10.3724/SP.J.1259.2014.00469 ZHANG Y N, ZHOU Y P, CHEN Q H, et al. Molecular basis of flowering time regulation in Arabidopsis [J]. Chinese Bulletin of Botany, 2014, 49(4): 469−482.(in Chinese) doi: 10.3724/SP.J.1259.2014.00469
[3]	KARLGREN A, GYLLENSTRAND N, KÄLLMAN T, et al. Evolution of the PEBP gene family in plants: Functional diversification in seed plant evolution [J]. Plant Physiology, 2011, 156(4): 1967−1977. doi: 10.1104/pp.111.176206
[4]	BANFIELD M J, BARKER J J, PERRY A C, et al. Function from structure? The crystal structure of human phosphatidylethanolamine-binding protein suggests a role in membrane signal transduction [J]. Structure, 1998, 6(10): 1245−1254. doi: 10.1016/S0969-2126(98)00125-7
[5]	VALVERDE F, MOURADOV A, SOPPE W, et al. Photoreceptor regulation of CONSTANS protein in photoperiodic flowering [J]. Science, 2004, 303(5660): 1003−1006. doi: 10.1126/science.1091761
[6]	张乔松, 杨伟儿. 中国水仙花芽分化及贮藏期外界因子对花序数的影响 [J]. 园艺学报, 1987, 14(2):139−143,145. ZHANG Q S, YANG W E. On flower-bud differentiation of Chinese narcissus and the effect of external factors in storage on flower percentage [J]. Acta Horticulturae Sinica, 1987, 14(2): 139−143,145.(in Chinese)
[7]	申艳红, 姜涛, 赵湾湾, 等. 乙烯处理水仙催多花技术和机理的研究 [J]. 农业生物技术学报, 2019, 27(6):1003−1015. SHEN Y H, JIANG T, ZHAO W W, et al. Study on technology and mechanism of ethylene treatment promotes the formation of more flowers of Narcissus tazetta var. chinensis [J]. Journal of Agricultural Biotechnology, 2019, 27(6): 1003−1015.(in Chinese)
[8]	ODA A, NARUMI T, LI T P, et al. CsFTL3, a chrysanthemum FLOWERING LOCUS T-like gene, is a key regulator of photoperiodic flowering in chrysanthemums [J]. Journal of Experimental Botany, 2012, 63(3): 1461−1477. doi: 10.1093/jxb/err387
[9]	MAO Y C, SUN J, CAO P P, et al. Functional analysis of alternative splicing of the FLOWERING LOCUS T orthologous gene in Chrysanthemum morifolium [J]. Horticulture Research, 2016, 3: 16058. doi: 10.1038/hortres.2016.58
[10]	WANG L J, SUN J, REN L P, et al. CmBBX8 accelerates flowering by targeting CmFTL1 directly in summer chrysanthemum [J]. Plant Biotechnology Journal, 2020, 18(7): 1562−1572. doi: 10.1111/pbi.13322
[11]	SUN J, WANG H, REN L P, et al. CmFTL2 is involved in the photoperiod- and sucrose-mediated control of flowering time in chrysanthemum [J]. Horticulture Research, 2017, 4: 17001. doi: 10.1038/hortres.2017.1
[12]	OTAGAKI S, OGAWA Y, HIBRAND-SAINT OYANT L, et al. Genotype of FLOWERING LOCUS T homologue contributes to flowering time differences in wild and cultivated roses [J]. Plant Biology, 2015, 17(4): 808−815. doi: 10.1111/plb.12299
[13]	CHEN L, CAI Y P, QU M N, et al. Soybean adaption to high-latitude regions is associated with natural variations of GmFT2b, an ortholog of FLOWERING LOCUS T [J]. Plant, Cell & Environment, 2020, 43(4): 934−944.
[14]	WU L, LI F, DENG Q H, et al. Identification and characterization of the FLOWERING LOCUS T/terminal flower 1 gene family in petunia [J]. DNA and Cell Biology, 2019, 38(9): 982−995. doi: 10.1089/dna.2019.4720
[15]	HELLER W P, YING Z T, DAVENPORT T L, et al. Identification of members of the Dimocarpus longan flowering locus T gene family with divergent functions in flowering [J]. Tropical Plant Biology, 2014, 7(1): 19−29. doi: 10.1007/s12042-013-9134-0
[16]	COELHO C P, MINOW M A A, CHALFUN-JÚNIOR A, et al. Putative sugarcane FT/TFL1 genes delay flowering time and alter reproductive architecture in Arabidopsis [J]. Frontiers in Plant Science, 2014, 5: 221.
[17]	NAVARRO C, ABELENDA J A, CRUZ-ORÓ E, et al. Control of flowering and storage organ formation in potato by FLOWERING LOCUS T [J]. Nature, 2011, 478(7367): 119−122. doi: 10.1038/nature10431
[18]	NIWA M, DAIMON Y, KUROTANI K I, et al. BRANCHED1 interacts with FLOWERING LOCUS T to repress the floral transition of the axillary meristems in Arabidopsis [J]. The Plant Cell, 2013, 25(4): 1228−1242. doi: 10.1105/tpc.112.109090
[19]	KINOSHITA T, ONO N, HAYASHI Y, et al. FLOWERING LOCUS T regulates stomatal opening [J]. Current Biology:CB, 2011, 21(14): 1232−1238. doi: 10.1016/j.cub.2011.06.025
[20]	CHEN M, PENFIELD S. Feedback regulation of COOLAIR expression controls seed dormancy and flowering time [J]. Science, 2018, 360(6392): 1014−1017. doi: 10.1126/science.aar7361
[21]	ANDRÉ D, MARCON A, LEE K C, et al. FLOWERING LOCUS T paralogs control the annual growth cycle in Populus trees [J]. Current Biology:CB, 2022, 32(13): 2988−2996.e4. doi: 10.1016/j.cub.2022.05.023
[22]	陈洁. 水稻FT-Like基因OsFTL4的功能研究[D]. 扬州: 扬州大学, 2020. CHEN J. Function research of FT-like gene OsTTL4 in rice [D]. Yangzhou: Yangzhou University, 2020. (in Chinese)
[23]	FENG Y, ZHU L Y, PAN T F, et al. Characterization of summer dormancy in Narcissus tazetta var. Chinensis and the role of NtFTs in summer dormancy and flower differentiation [J]. Scientia Horticulturae, 2015, 183: 109−117. doi: 10.1016/j.scienta.2014.11.013
[24]	LI X F, JIA L Y, XU J, et al. FT-like NFT1 gene may play a role in flower transition induced by heat accumulation in Narcissus tazetta var. chinensis [J]. Plant and Cell Physiology, 2013, 54(2): 270−281. doi: 10.1093/pcp/pcs181
[25]	CONANT G C, WOLFE K H. Turning a hobby into a job: How duplicated genes find new functions [J]. Nature Reviews Genetics, 2008, 9(12): 938−950. doi: 10.1038/nrg2482
[26]	李永光, 金玉环, 郭力, 等. 小鼠耳芥PEBP基因家族全基因组鉴定及表达分析 [J]. 遗传, 2022, 44(1):80−94. LI Y G, JIN Y H, GUO L, et al. Genome-wide identification and expression analysis of the PEBP genes in Arabidopsis pumila [J]. Hereditas(Beijing), 2022, 44(1): 80−94.(in Chinese)
[27]	牛西强, 罗潇云, 康凯程, 等. 辣椒PEBP基因家族的全基因组鉴定、比较进化与组织表达分析 [J]. 园艺学报, 2021, 48(5):947−959. NIU X Q, LUO X Y, KANG K C, et al. Genome-wide identification, comparative evolution and expression analysis of PEBP gene family from Capsicum annuum [J]. Acta Horticulturae Sinica, 2021, 48(5): 947−959.(in Chinese)
[28]	JIANG X D, ZHONG M C, DONG X, et al. Rosoideae-specific duplication and functional diversification of FT-like genes in Rosaceae [J]. Horticulture Research, 2022, 9: uhac059. doi: 10.1093/hr/uhac059
[29]	LIU H L, LIU X, CHANG X J, et al. Large-scale analyses of angiosperm Flowering Locus T genes reveal duplication and functional divergence in monocots [J]. Frontiers in Plant Science, 2023, 13: 1039500. doi: 10.3389/fpls.2022.1039500
[30]	LEEGGANGERS H A, ROSILIO-BRAMI T, BIGAS-NADAL J, et al. Tulipa gesneriana and Lilium longiflorum PEBP genes and their putative roles in flowering time control [J]. Plant and Cell Physiology, 2018, 59(1): 90−106. doi: 10.1093/pcp/pcx164
[31]	LEE R, BALDWIN S, KENEL F, et al. FLOWERING LOCUS T genes control onion bulb formation and flowering [J]. Nature Communications, 2013, 4: 2884. doi: 10.1038/ncomms3884
[32]	YAN X, CAO Q Z, HE H B, et al. Functional analysis and expression patterns of members of the FLOWERING LOCUS T (FT) gene family in Lilium [J]. Plant Physiology and Biochemistry, 2021, 163: 250−260. doi: 10.1016/j.plaphy.2021.03.056
[33]	KOTODA N, HAYASHI H, SUZUKI M, et al. Molecular characterization of FLOWERING LOCUS T-like genes of apple (Malus ×domestica borkh. ) [J]. Plant and Cell Physiology, 2010, 51(4): 561−575. doi: 10.1093/pcp/pcq021
[34]	朱燕宇. 小叶杨FT基因家族的克隆及功能验证[D]. 南京: 南京林业大学, 2015. ZHU Y Y. Cloning and functional analysis of FT gene family from Populus simonii[D]. Nanjing: Nanjing Forestry University, 2015. (in Chinese)
[35]	PIN P A, NILSSON O. The multifaceted roles of FLOWERING LOCUS T in plant development [J]. Plant, Cell & Environment, 2012, 35(10): 1742−1755.
[36]	LIU W, JIANG B J, MA L M, et al. Functional diversification of Flowering Locus T homologs in soybean: GmFT1a and GmFT2a/5a have opposite roles in controlling flowering and maturation [J]. The New Phytologist, 2018, 217(3): 1335−1345. doi: 10.1111/nph.14884