Cloning and Expression Analysis of PoFD Gene from Paeonia ostii ‘Fengdan’

doi:10.13560/j.cnki.biotech.bull.1985.2022-0004

Abstract

Abstract:

Flowering Locus D（FD）gene, a member of the bZIP transcription factor family, plays an important role in promoting plant flowering interacting with the FT gene via interaction with FT gene. In this study, tree peony（Paeonia ostii）variety ‘Fengdan’ was used as the material, the PoFD gene was cloned by RT-qPCR technology based on the results of the three-generation full-length transcriptome sequencing of ‘Fengdan’, and the bioinformatics and expression patterns analysis were performed on it. The results showed that the cloned PoFD gene contained a 2 712 bp complete ORF frame, encoding 903 amino acids. The molecular formula of PoFD protein is C₈₂₆₁H₁₃₇₂₂N₂₇₁₂O₃₄₆₈S₅₅₂, and the theoretical isoelectric point（pI）is 4.88. It is a hydrophilic protein and has no transmembrane structure. There are a high proportion of random coils and α-helices in secondary structure, while β-turns account for only a small part. RT-qPCR analysis demonstrated the expression of PoFD gene was the highest in leaves, thus speculating that PoFD gene may mainly function in leaves to regulate the flowering stage of tree peony. In different flower development stages, the expression of PoFD was the highest at the half-open stage, inferring that the PoFD gene may play the role at the middle flowering stage. Compared with the control group, the flowering stage of ‘Fengdan’ delayed at varying degrees while treated with varied concentrations of brassinosteroid（BR）, and the expression of PoFD gene decreased, indicating that PoFD gene may regulate tree peony flowering period in response to BR. This study provides a theoretical reference for the follow-up study of the role of PoFD gene in the regulation of tree peony flowering.

Key words: Paeonia ostii ‘Fengdan’, flowering regulating, PoFD, gene cloning, bioinformatics analysis, expression analysis

ZHANG Lin, WEI Zhen-zhen, SONG Cheng-wei, GUO Li-li, GUO Qi, HOU Xiao-gai, WANG Hua-fang. Cloning and Expression Analysis of PoFD Gene from Paeonia ostii ‘Fengdan’[J]. Biotechnology Bulletin, 2022, 38(11): 104-111.

Figures/Tables 9

Table 1 Primer sequence

引物名称 Primer name	序列 Sequence（5'-3'）	扩增目的 Amplification purpose
PoFD-F	TTTACCGGACCTGAACAC	克隆PoFD基因 Cloning of PoFD gene
PoFD-R	ACCCAACACCCTCATACA	克隆PoFD基因 Cloning of PoFD gene
PoFD-RT-F	ATTGTAGGCGGTAGGAGCAC	PoFD基因RT-qPCR分析 RT-qPCR assay of PoFD gene
PoFD-RT-R	TATCAAGCACGGTCGCTGT	PoFD基因RT-qPCR分析 RT-qPCR assay of PoFD gene
Actin-F Actin-R	GGTCTATTCTTGCTTCCCTCAG GAACTCACTATCAAACCCTCCAG	RT-qPCR内参基因 RT-qPCR reference gene

Fig. 1 Electrophoresis diagram of ‘Fengdan’ PoFD gene M：DL5000 DNA marker. 1：Amplified product of PoFD gene

Fig. 2 Analysis of phosphorylation sites in ‘Fengdan’ PoFD protein

Fig. 3 Secondary structure prediction of PoFD protein

Fig. 4 Tertiary structure prediction of PoFD protein

Fig. 5 Phylogenetic tree analysis of ‘Fengdan’ PoFD protein

Fig. 6 Expression analysis of PoFD gene in different tissues of ‘Fengdan’ Different lowercases indicate significant differences at the 0.05 level. The same below

Fig. 7 Analysis of expression of PoFD gene in different flower development stages of ‘Fengdan’

Fig. 8 Expression analysis of PoFD gene under different concentrations of BR hormone treatment

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