Cloning, Differential Expression and Subcellular Localization of FeTOC1 Gene in Tall Fescue

doi:10.11869/j.issn.100-8551.2022.02.0302

Journal of Nuclear Agricultural Sciences ›› 2022, Vol. 36 ›› Issue (2): 302-312.DOI: 10.11869/j.issn.100-8551.2022.02.0302

• Induced Mutations for Plant Breeding·Agricultural Biotechnology • Previous Articles Next Articles

Cloning, Differential Expression and Subcellular Localization of FeTOC1 Gene in Tall Fescue

WANG Qian¹(), WU Jiahai², CHEN Ying¹, WANG Xiaoli¹^,^*()

¹Guizhou Institute of Prataculture, Guizhou Academy of Agricultural Sciences, Guiyang, Guizhou 550006
²Guizhou Institute of Fruit Tree Science, Guizhou Academy of Agricultural Sciences, Guiyang, Guizhou 550006

Received:2020-11-18 Accepted:2021-01-07 Online:2022-02-10 Published:2022-01-17
Contact: WANG Xiaoli

高羊茅FeTOC1基因的克隆、差异表达及亚细胞定位分析

王茜¹(), 吴佳海², 陈莹¹, 王小利¹^,^*()

¹贵州省农业科学院草业研究所,贵州贵阳 550006
²贵州省农业科学院果树研究所,贵州贵阳 550006

通讯作者: 王小利
作者简介:王茜,女,主要从事牧草逆境生理及分子生物学研究。E-mail: snoopy0729@163.com
基金资助:
国家自然科学基金项目(31802128);贵州省高层次创新人才培养项目(黔科合平台人才(2018)5634);贵州省科技计划项目(黔科合平台人才(2020)5005);贵州省科技计划(黔科合基础[2020]1Z027)

Abstract

Abstract:

TOC1 gene is a major component of the central oscillator, which regulates the response to illumination through photoperiod pathway in Arabidopsis thaliana. In order to illustrate the biological function of FeTOC1 in Festuca elata, we obtained the full-length cDNA sequence of FeTOC1 by homologous cloning, analyzed the expression patterns of FeTOC1 under diverse light levels, and conducted the subcellular localization of FeTOC1. The results indicated that the full-length of FeTOC1 was 1 557 bp, encoding 517 amino acid residues; the ended protein FeTOC1 contained the pseudo-receiver domain of about 120 amino acids at N-terminus and the short CCT motif of about 50 amino acids at C-terminus; the phylogenetic tree analysis showed that FeTOC1 had close relations to those in barley, wheat and coarse goat grass, which were up to 99%. Then, the constructed target protein was fused with the GFP expression vector, and injected into tobacco, the expression suggested that FeTOC1 was mainly located in the nucleus. Real-time fluorescence quantitative analysis of the expression patterns of FeTOC1 in leaves revealed that the expression of FeTOC1 had a diurnal rhythm under both long- and short-day, and the expression at illumination 0~4 h reached the highest; compared with continuous darkness, continuous illumination weakened the oscillatory rhythm of FeTOC1 dramatically; reversing the circadian rhythm, FeTOC1 was preferentially regulated by environmental signal. To sum up, FeTOC1 gene played an important role in circadian clock regulation in tall fescue.

Key words: Festuca elata (tall fescue), TOC1 gene, expression pattern, subcellular localization

摘要：

拟南芥TOC1基因是中央振荡器的重要组成部分,其编码的蛋白质TOC1通过光周期途径调控拟南芥对光照的响应。为了揭示高羊茅FeTOC1基因的生物学功能,本研究通过同源克隆获得该基因全长cDNA序列,对其在不同光照水平下的表达模式进行分析,并对其编码的蛋白质进行亚细胞定位。结果显示,高羊茅FeTOC1基因的全长cDNA序列为1 557 bp,编码517个氨基酸; 其编码的蛋白质N端存在大约120个氨基酸组成的非典型PR结构域,C端则存在大约50个氨基酸构成的CCT结构域; 系统进化树分析显示高羊茅FeTOC1蛋白质与禾本科大麦、小麦及粗山羊草亲缘关系高达99%。将构建的目的蛋白与GFP融合表达载体注射入烟草中,发现FeTOC1主要定位在细胞核中。实时荧光定量分析发现,叶片中FeTOC1表达在长、短日照下表现出一定的昼夜节律性,表达峰值均出现在光照0~4 h;相比于持续黑暗,持续光照显著地削弱了FeTOC1表达的振荡节律;颠倒昼夜节律,FeTOC1基因的表达首先受外界昼夜交替信号的调节。综上所述,FeTOC1基因在高羊茅生物钟调节中发挥着重要作用。本研究结果为进一步探索FeTOC1基因的功能,进而培育适应短日照地区牧草新品种提供了一定的理论基础。

关键词: 高羊茅, TOC1基因, 表达模式, 亚细胞定位

WANG Qian, WU Jiahai, CHEN Ying, WANG Xiaoli. Cloning, Differential Expression and Subcellular Localization of FeTOC1 Gene in Tall Fescue[J]. Journal of Nuclear Agricultural Sciences, 2022, 36(2): 302-312.

王茜, 吴佳海, 陈莹, 王小利. 高羊茅FeTOC1基因的克隆、差异表达及亚细胞定位分析[J]. 核农学报, 2022, 36(2): 302-312.

Figures/Tables 10

Table 1 Treatments of diverse photoperiod patterns

处理 Treatments	光周期模式及处理时间 Photoperiod patterns and treatment time
处理一 The first treatment	长日照/4周
处理一 The first treatment	短日照/4周
处理二 The second treatment	长日照/3周	连续光照/1周
	长日照/3周	连续黑暗/1周
	短日照/3周	连续光照/1周
	短日照/3周	连续黑暗/1周
处理三 The third treatment	遵循外界昼夜长日照/3周	继续遵循/1周
	遵循外界昼夜长日照/3周	颠倒外界昼夜/1周
	颠倒外界昼夜长日照/3周	继续颠倒/1周
	颠倒外界昼夜长日照/3周	遵循外界昼夜/1周

Table 2 Sequences of primers used for cloning and qRT-PCR amplification of FeTOC1

引物名称Primer	引物序列 (5'→3') Primer sequence (5'→3')	扩增类别 Amplification sort
TOC1F2	AAGTGCTTCAAGATGCTCAA	核心片段
TOC1R2	CTRAAYTTKGCAAGTGCTGC
C341-1	TGTATCTACGATGCCGTCATTTCA	3'端快速扩增
C341-2	ACATTCAAGTCATTTGTCAACGCA
B463-1(GSP1)	CGGCTTGACCAGGTAC	5'端快速扩增
B463-2(GSP2)	ACAACAACAGAAACATCG
B463-3(GSP3)	TGATGATGGGGATGTGGC
TOC1-F	GGCCAAAAGAAAACAAAAAT	qRT-PCR
TOC1-R	GACGACTCAGCAAACCTCAA
GAPDH-F	ACCCCTTCATCACCACCGACTAC	内参基因
GAPDH-R	TCCTTCTCGTTGACACCCATGAC

Fig.1 Agarose gel electrophoresis of semi-quantitative reverse transcription-PCR products

Fig.2 Nucleotide and deduced amino acid sequence of FeTOC1 from Festuca elata

Fig.3 Amino acid sequence alignment of FeTOC1 from Festuca elata with others from hight plants Note: The red and green underlined regions represent the conserved PR and CCT domains, respectively.

Fig.4 Phylogenetic tree of FeTOC1 from Festuca elata and those from other plants

Fig.5 Subcellular localization of FeTOC1

Fig.6 FeTOC1 gene expression level under long-(A) and short-days (B) in Festuca elata leaves Note: The black bar on the horizontal axis represents the dark treatment. The white bars represent light treatment. The same as following.

Fig.7 FeTOC1 gene expression level under continual illumination (A and C) and continual darkness (B and D) in Festuca elata leaves after long-(A and B) and short-days (C and D)

Fig.8 FeTOC1 gene expression level under day and night inversion in Festuca elata leaves

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创刊：1987年
主管部门：农业农村部
主办单位：中国原子能农学会与中国农业科学院农产品加工研究所（前中国农业科学院原子能利用研究所）
ISSN：1000-8551

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Cloning, Differential Expression and Subcellular Localization of FeTOC1 Gene in Tall Fescue

高羊茅FeTOC1基因的克隆、差异表达及亚细胞定位分析

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