Expression,Protein Interactions and Biological Function Analysis of FaFT in Festuca arundinacea

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

Abstract

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

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.

Figures/Tables 10

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

Fig. 1 Nucleotide sequence and amino acid sequence of FaFT

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

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

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)

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)

Fig.6 FaFT expression in tobacco epidermal cells

Fig.7 Screening of interaction between FaFT and FD

Fig. 8 BiFC experimental verification of FaFT and FD

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

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