The SEPALLATA-like gene HrSEP1 in Hippophae rhamnoides regulates flower development by interacting with other MADS-box subfamily genes

Abstract

MADS-box genes are classified into five categories: ABCDE, including SEP1, SEP2, SEP3, SEP4, and other homologous genes, which play important roles in floral organ development. In this study, the cDNA sequence of the HrSEP1 gene was cloned by RT-PCR and confirmed that this gene belongs to the MADS-box gene family. In addition, subcellular localization experiments showed that the HrSEP1 protein was localized in the nucleus. We verified the interaction of HrSEP1 with HrSOC1, HrSVP, and HrAP1 using yeast two-hybrid and bimolecular fluorescence complementation assays. These genes jointly regulate the growth and development of floral organs. We also found a strong synergy between HrSEP1 and AP1 genes in sepals, petals, and stamens by transgenic methods and fluorescence quantitative PCR, suggesting that HrSEP1 and AP1 may co-regulate the development of these structures. In conclusion, the expression of HrSEP1 has a certain effect on the development of floral organs, and these findings lay the foundation for further research on the biological functions of MADS transcription factors in Hippophae rhamnoides.

Keywords: Hippophae rhamnoides; MADS-box genes; bimolecular fluorescence complementation; genetic transformation; yeast two-hybrid.

Figure 1

Phylogenetic tree of the MADS-box gene in Hippophae rhamnoides L. and the MADS-box genes in Arabidopsis thaliana, Actinidia chinensis Planch, Hevea brasiliensis, and Solanum lycopersicum L. Arabidopsis gene sequences from The Arabidopsis Information Resource (TAIR) database; other gene sequences were obtained from the National Center for Biotechnology Information (NCBI) database. Different subfamilies were marked with specific colors. Trees were constructed using the neighbor-joining method, and 1,000 bootstrap replications were performed using MEGA 7.0 International software.

Figure 2

Subcellular localization of HrSEP1. Combining HrSEP1 gene with fluorescent protein vector pCAMBIA1300-GFP can determine the target gene expression location. Green fluorescence protein (GFP) as nuclear marker.

Figure 3

Plant expression vectors were constructed, and T-HrSEP1 genomic DNA was detected after genetic transformation of tobacco to verify the successful transfer of the HrSEP1 gene into tobacco. (A) Schematic diagram of plant overexpression vectors. (B) Transgenic tobacco was obtained using the leaf disc method; from left to right, co-cultures, screening cultures, and differentiation cultures. (C) Detection of T-HrSEP1 genomic DNA in the T-HrSEP1 genome by PCR: M, DL2000Marker; 1–18, T-HrSEP1 genomic DNA. HrSEP1 gene: M, DL2000Marker; 1–18, T-HrSEP1 genomic DNA; −, negative control; +, positive control.

Figure 4

Expression analysis of floral development-related genes HrSEP1, NcSOC1, NcSVP, and NcAP1 in different parts of floral tissues (including petals, sepals, pistils, and stamens) in transgenic tobacco. Significant differences between each group of data were determined by Student’s t-test (**: 0.001 < p < 0.01; ***: 0.0001 < p < 0.001; ****: 0.00001 < p < 0.0001).

Figure 5

Phenotypic observation of transgenic plants. The scale has a score of 1 cm. (A) Comparison of petal shape and color between transgenic tobacco and wild tobacco. (B) Comparison of stamen size between transgenic tobacco and wild tobacco. (C) Comparison of petal diameters. (D) Comparison of stamen lengths. Significant differences between treatment means were determined by Student’s t-test (*, p < 0.05).

Figure 6

Interaction between HrSEP1 and HrAP1, HrSOC1, and HrSVP in yeast-competent cells. The interaction is indicated by the growth of blue plaque on SD/Leu/-Trp/-His/-Ade/nutrient-deficient medium. (A) Yeast self-activation experiment verifies that HrSEP1 does not have self-activation activity. (B) Yeast two-hybrid experiment to verify the interaction between HrSEP1 and three other genes.

Figure 7

The fluorescence expression of three interaction combinations in Nicotiana benthamiana leaves was detected by bimolecular fluorescence complementary experiment to clarify the interaction relationship. Yellow fluorescent protein (YFP) as nuclear marker.