高羊茅FaFT基因表达、蛋白互作及生物学功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-0927

摘要/Abstract

摘要：

FLOWERING LOCUS T（FT）对植物开花有重要的调控作用。为探索高羊茅FaFT基因的生物学功能,从前期高羊茅转录组中克隆得到FaFT基因。qRT-PCR分析表明FaFT基因在长日照、短日照、连续光照、连续黑暗、昼夜颠倒和长日照下不同发育阶段都保持一定的昼夜节律并受光周期的调控。亚细胞定位显示FaFT基因编码的蛋白位于细胞核中;利用酵母双杂交筛选出可能与FaFT互作的FD蛋白,并进一步通过双分子荧光互补（BiFC）方法进行验证FaFT与FD蛋白互作的真实性。通过构建过表达载体和功能补偿发现在长日照条件下FaFT基因促进拟南芥提前开花,FT基因功能补偿后恢复拟南芥突变体开花表型,突变体植株本身没有开花现象。以上结果说明高羊茅FaFT基因的生物学功能得以成功验证。

关键词: 高羊茅, FT基因, 蛋白互作, 生物功能

Abstract:

FLOWERING LOCUS T（FT）plays a significant role in regulating plant flowering. In this study,the FaFT gene was cloned from the previous tall fescue’s transcriptome in order to explore the FaFT gene biological function. qRT-PCR analysis showed that the FaFT gene maintained a certain circadian rhythm and was regulated by the light cycle in different developmental stages under long-day,short-day,continuous light,continuous darkness,day-night inversion,and long-day light. Subcellular localization revealed that the FaFT protein was located in the nucleus. FD（FLOWERING LOCUS D）protein that may interact with FaFT was selected by yeast two-hybrid,and the authenticity of the interaction between FaFT and FD protein was further verified by the bimolecular fluorescence complementation（BiFC）method. Through the construction of overexpression vectors and functional compensation,it was found that FaFT gene promoted Arabidopsis early flowering under long-day condition. After functional compensation of FT gene,the flowering phenotype of Arabidopsis mutants was restored,and the mutant plants themselves showed no flowering phenomenon. The above results indicate that the biological function of the FaFT gene of F. arundinacea has been successfully verified.

Key words: Festuca arundinacea, FT gene, protein interaction, biological function

罗维, 牟琼, 舒健虹, 吴佳海, 王小利. 高羊茅FaFT基因表达、蛋白互作及生物学功能分析[J]. 生物技术通报, 2021, 37(4): 8-17.

LUO Wei, MU Qiong, SHU Jian-hong, WU Jia-hai, WANG Xiao-li. Expression,Protein Interactions and Biological Function Analysis of FaFT in Festuca arundinacea[J]. Biotechnology Bulletin, 2021, 37(4): 8-17.

图/表 10

表1 引物信息

Table 1 Primer sequence

引物Primer	序列Sequence（5'-3'）	用途Purpose
FaFT-F1	GCGGGAACCGTTGTCTCTAT	基因组扩增Genome amplification
FaFT-R1	GTCCGGTCACGACCTTCAAT	基因组扩增Genome amplification
FaFT-F2	GGATCCTTTGGCATTGAGCC	实时荧光定量PCR Real-time fluorescence quantitative PCR
FaFT-R2	CCGGTCACGACCTTCAATCT	实时荧光定量PCR Real-time fluorescence quantitative PCR
FaGAPDH-F	ACCCCTTCATCACCACCGACTAC	内参基因Reference gene
FaGAPDH-R	TCCTTCTCGTTGACACCCATGAC	内参基因Reference gene
FaFT-BD-F	GCATATGGCCATGGAGGCCGAATTC	酵母双杂交Yeast two-hybrid
FaFT-BD-R	GCGGCCGCTGCAGGTCGACGGATCC
FaFD-AD-F	TATGGCCATGGAGGCCAGTGAATTC
FaFD-AD-R	TCTGCAGCTCGAGCTCGATGGATCC
FaFT-C-F	GCCTGGCGCGCCACTAGTGGATCCA	双分子荧光互补Bimolecular fluorescence complementation
FaFT-C-R	CATCCCGGGAGCGGTACCCTCGAG	双分子荧光互补Bimolecular fluorescence complementation
Primer F	GGGGTACCATGTCGCGGGGCAGGGAT	过表达载体构建Overexpression vector construction
Primer R	GCTCTAGATTAGCTTGTTCCTTGGAA	过表达载体构建Overexpression vector construction

图1 FaFT基因的核苷酸序列及氨基酸序列

Fig. 1 Nucleotide sequence and amino acid sequence of FaFT

图2 长日照（A）、短日照（B）下高羊茅FaFT基因的表达水平

Fig. 2 Expression level of FaFT gene in tall fescue under long day (A) and short day (B)

图3 长日照转连续光照（A）和连续黑暗（B）、短日照转连续光照（C）和连续黑暗（D）高羊茅FaFT基因的表达水平

Fig.3 Expression level of FaFT gene of tall fescue from sunlight to continuous light (A) and continuous darkness (B) based on long day, continuous light (C) and continuous darkness (D) based on short day

图4 高羊茅在光照/黑暗（A）、光照/黑暗-黑暗/光照（B）、黑暗/光照（C）、黑暗/光照-光照/黑暗（D）下FaFT基因的表达水平

Fig.4 Expression level of FaFT gene in tall fescue under light/dark (A), light/dark-dark/light (B), dark/light (C), dark/light-light/dark (D)

图5 高羊茅FaFT基因在苗期（A）、分蘖期（B）、孕穗期（C）、抽穗期（D）的表达分析

Fig. 5 Expression analysis of the FaFT gene of tall fescue at the seedling stage (A), tillering stage (B), booting stage (C) and heading stage (D)

图6 FaFT在烟草表皮细胞中的表达

Fig.6 FaFT expression in tobacco epidermal cells

图7 FaFT与FD相互作用的筛选

Fig.7 Screening of interaction between FaFT and FD

图8 FaFT与FD的BiFC实验验证

Fig. 8 BiFC experimental verification of FaFT and FD

图9 转基因拟南芥的开花时间、莲座叶数量及表型

Fig.9 Flowering time, rosette leaf number and phenotype of transgenic Arabidopsis

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