毛竹PheFT1基因的表达、蛋白互作及生物学功能分析

doi:10.11707/j.1001-7488.LYKX20240069

摘要/Abstract

摘要：

目的: 分析毛竹PheFT1基因的表达、蛋白互作及生物学功能，为揭示该基因调控开花机理提供理论依据。方法: 利用实时荧光定量PCR检测PheFT1的组织特异性表达及对光周期的应答反应；采用PEG介导法分析PheFT1蛋白定位；应用农杆菌介导法将PheFT1基因转入拟南芥获得过表达植株及ft突变体回补植株，比较过表达植株与野生型植株、ft突变体回补植株与ft突变体植株之间的表型差异分析其生物学功能；基于酵母双杂交和双分子荧光互补试验分析PheFT1的互作蛋白。结果: 生物信息学分析结果显示，PheFT1基因CDS全长537 bp，编码178个氨基酸，属PEBP蛋白家族。亚细胞定位结果显示，PheFT1蛋白定位于细胞核和细胞质。实时荧光定量PCR结果显示，PheFT1基因在根、茎、叶和侧芽中均有表达，在侧芽和茎中表达量较高，在叶中表达量最低；长日照下PheFT1有较强的昼夜节律。异位表达结果显示，PheFT1基因会使拟南芥开花提前、茎变细和株高变矮。蛋白互作结果显示，PheFT1可与PheGF14和PheFD蛋白互作。结论: 毛竹PheFT1不仅是开花促进因子，还参与茎秆发育和高生长，该研究为进一步揭示PheFT1基因参与毛竹开花的分子机制及生长发育提供了参考。

关键词: 毛竹, FT基因, PEBP蛋白家族, 蛋白互作, 开花

Abstract:

Objective: OsFTL1 in rice is a flowering regulator which can also affect leaf size, plant height and spike structure. At the early stage, 18 FT genes were identified from Phyllostachys edulis (moso bamboo), one of which exhibited the closest genetic relationship with OsFTL1. This study aims to clarify the biological function of this gene in moso bamboo in order to provide a theoretical basis for revealing the mechanism of flowering in moso bamboo. Method: Real-time quantitative PCR was used to detect the tissue-specific expression of PheFT1 and its response to photoperiod. The subcellular localization of PheFT1 protein was analyzed by PEG-mediated assay. Agrobacterium-mediated transfer of PheFT1 gene into Arabidopsis thaliana was used to produce overexpression plants and ft mutant complemented plants. The phenotypic differences between overexpression plants and wild-type plants as well the differences between ft mutant complementation plants and ft mutant plants were compared to analyze PheFT1 biological function. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation assays were used to analyze the interacting proteins of PheFT1. Result: The results of bioinformatics analysis showed that the CDS of PheFT1 gene was 537 bp in full length, encoding 178 amino acids, belonging to the PEBP protein family. The results of subcellular localization showed that PheFT1 protein was localized in the nucleus and cytoplasm. The real-time quantitative PCR showed that the PheFT1 gene was expressed in root, stem, leaf and lateral bud, with higher expression in lateral bud and stem, and lower expression in leaf. PheFT1 exhibited a strong circadian rhythm under long-day. Ectopic expression showed that the PheFT1 gene induced earlier flowering, thinner stem and shorter plant height in A. thaliana. Moreover, the results of protein interaction showed that PheFT1 was able to interact with PheGF14 and PheFD proteins. Conclusion: PheFT1 is a flowering promoting factor in moso bamboo, and also participates in the stem development and height growth. This study provides a reference for further revealing the molecular mechanism of the PheFT1 gene involved in flowering and development in moso bamboo.

Key words: Phyllostachys edulis, FLOWERING LOCUS T gene, PEBP protein family, protein interaction, flowering

中图分类号:

S795.7

闫小玲,郝琴,申孜,张雨佳,郭小勤. 毛竹PheFT1基因的表达、蛋白互作及生物学功能分析[J]. 林业科学, 2025, 61(4): 140-152.

Xiaoling Yan,Qin Hao,Zi Shen,Yujia Zhang,Xiaoqin Guo. Expression, Protein Interaction and Biological Function Analysis of PheFT1 Gene in Moso Bamboo[J]. Scientia Silvae Sinicae, 2025, 61(4): 140-152.

图/表 9

表1

本研究中使用的 PCR 引物"

引物名称 Primer name	引物序列（5'–3'） Primer sequence (5'–3')	引物用途 Application
PheFT1-F	ATGGCCAGCGGCAGCGTG	基因克隆
PheFT1-R	TCACATTCTTCTCCCACCAGTTCCCGA	Gene cloning

PheFT1-pCAMBIA1300-F	GAGCTCGGTACCCGGGGATCCATGGCCAGCGGCAGCGTG	构建亚细胞定位载体
PheFT1-pCAMBIA1300-R	CATGTCGACTCTAGAGGATCCCATTCTTCTCCCACCAGTTCCCGA	Construction of subcellular localization plasmid ppplasmidplasmids

PheFT1-QF	GGTGATCGGCGACGTGGT
PheFT1-QR	TGTAGAATGTGCGCATGTCC	荧光定量 PCR
PheTIP41-QF	AAAATCATTGTAGGCCATTGTCG	Quantitative real time PCR
PheTIP41-QR	ACTAAATTAAGCCAGCGGGAGTG

PheFT1-pCAMBIA1300s-F	GGTACCCGGGGATCCTCTAGAATGGCCAGCGGCAGCGTG	构建过表达载体
PheFT1-pCAMBIA1300s-R	CTTGCATGCCTGCAGGTCGACTCACATTCTTCTCCCACCAGTTCCCGA	Construction of overexpression plasmid

PheFT1-pGADT7-F	GAGTGGCCATTATGGCCCGGGATGGCCAGCGGCAGCGTG
PheFT1-pGADT7-R	GCCGACATGTTTTTTCCCGGG TCACATTCTTCTCCCACCAGTTCCCGA
PheFT1-pGBKT7-F	GGCCGAATTCCCGGGGATGGCCAGCGGCAGCGTG
PheFT1-pGBKT7-R	CCGCTGCAGGTCGACGGATCC TCACATTCTTCTCCCACCAGTTCCCGA
PheGF14a-pGADT7-F	GAGTGGCCATTATGGCCCGGGATGGGTACGGCAGCGGGA
PheGF14a-pGADT7-R	GCCGACATGTTTTTTCCCGGGCTAGTGCTCATCCTCGGGCTT
PheGF14b-pGADT7-F	GAGTGGCCATTATGGCCCGGGATGTCGGCACCTGCGGAG
PheGF14b-pGADT7-R	GCCGACATGTTTTTTCCCGGGTTATCCAGAGCAAACAGC
PheGF14c-pGADT7-F	GAGTGGCCATTATGGCCCGGGATGTCGCGGGAGGAGAATG
PheGF14c-pGADT7-R	GCCGACATGTTTTTTCCCGGGTCACTGGCCCTCGCCGGC	构建酵母杂交载体
PheGF14d-pGADT7-F	GAGTGGCCATTATGGCCCGGGATGTCGCCGGCGGAGCCG	Construction of yeast two-hybrid plasmids
PheGF14d-pGADT7-R	GCCGACATGTTTTTTCCCGGGTCACAGTCCATCTCCGGATTCC
PheGF14e-pGADT7-F	GAGTGGCCATTATGGCCCGGGATGTCGCAGCCTGCCGAG
PheGF14e-pGADT7-R	GCCGACATGTTTTTTCCCGGGTTACTGCCCATCTCCAGATTCG
PheGF14f-pGADT7-F	GAGTGGCCATTATGGCCCGGGATGTCTACTGCTGAAGCATCCCA
PheGF14f-pGADT7-R	GCCGACATGTTTTTTCCCGGGTTAGTGCCCCTCTCCTTCAGG
PheGF14g-pGADT7-F	GAGTGGCCATTATGGCCCGGGATGGCGTCGCGCGAGGAT
PheGF14g-pGADT7-R	GCCGACATGTTTTTTCCCGGGTCAACCAGATATACGAGCATCAGC
PheGF14h-pGADT7-F	GAGTGGCCATTATGGCCCGGGATGGAGGAGAGGGAGAAGGTCG
PheGF14h-pGADT7-R	GCCGACATGTTTTTTCCCGGGCTAGAAACCTCCAGTTAGTTCTGACG
PheFD-pGADT7-F	GAGTGGCCATTATGGCCCGGGATGGCCAACTACCACCACTA
PheFD-pGADT7-R	GCCGACATGTTTTTTCCCGGGTCATCCTCTTAATTTTGTACTGCCA

PheFT1-pSAT1-F	AGATCTCGAGCTCAAGCTTCGATGGCCAGCGGCAGCGTG
PheFT1-pSAT1-R	GGTACCGTCGACTGCAGAATTTCACATTCTTCTCCCACCAGTTCCCGA
PheFT1-pSAT4-F	AGATCTCGAGCTCAAGCTTCGATGGCCAGCGGCAGCGTG
PheFT1-pSAT4-R	GGTACCGTCGACTGCAGAATTTCACATTCTTCTCCCACCAGTTCCCGA
PheGF14a-pSAT4-F	AGATCTCGAGCTCAAGCTTCGATGGGTACGGCAGCGGGA
PheGF14a-pSAT4-R	GGTACCGTCGACTGCAGAATTCTAGTGCTCATCCTCGGGCTT
PheGF14b-pSAT4-F	AGATCTCGAGCTCAAGCTTCGATGTCGGCACCTGCGGAG
PheGF14b-pSAT4-R	GGTACCGTCGACTGCAGAATTTTATCCAGAGCAAACAGC
PheGF14c-pSAT4-F	AGATCTCGAGCTCAAGCTTCGATGTCGCGGGAGGAGAATG
PheGF14c-pSAT4-R	GGTACCGTCGACTGCAGAATTTCACTGGCCCTCGCCGGC	构建BiFC载体
PheGF14d-pSAT4-F	AGATCTCGAGCTCAAGCTTCGATGTCGCCGGCGGAGCCG	Construction of BiFC plasmids
PheGF14d-pSAT4-R	GGTACCGTCGACTGCAGAATTTCACAGTCCATCTCCGGATTCC
PheGF14e-pSAT4-F	AGATCTCGAGCTCAAGCTTCGATGTCGCAGCCTGCCGAG
PheGF14e-pSAT4-R	GGTACCGTCGACTGCAGAATTTTACTGCCCATCTCCAGATTCG
PheGF14f-pSAT4-F	AGATCTCGAGCTCAAGCTTCGATGTCTACTGCTGAAGCATCCCA
PheGF14f-pSAT4-R	GGTACCGTCGACTGCAGAATTTTAGTGCCCCTCTCCTTCAGG
PheGF14g-pSAT4-F	AGATCTCGAGCTCAAGCTTCGATGGCGTCGCGCGAGGAT
PheGF14g-pSAT4-R	GGTACCGTCGACTGCAGAATTTCAACCAGATATACGAGCATCAGC
PheGF14h-pSAT4-F	AGATCTCGAGCTCAAGCTTCGATGGAGGAGAGGGAGAAGGTCG
PheGF14h-pSAT4-R	GGTACCGTCGACTGCAGAATTCTAGAAACCTCCAGTTAGTTCTGACG
PheFD-pSAT4-F	AGATCTCGAGCTCAAGCTTCGATGGCCAACTACCACCACTA
PheFD-pSAT4-R	GGTACCGTCGACTGCAGAATTTCATCCTCTTAATTTTGTACTGCCA

表1

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