Molecular Cloning, Characterization, and Expression of MiSOC1: A Homolog of the Flowering Gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 from Mango (Mangifera indica L)

Abstract

MADS-box transcription factor plays a crucial role in plant development, especially controlling the formation and development of floral organs. Mango (Mangifera indica L) is an economically important fruit crop, but its molecular control of flowering is largely unknown. To better understand the molecular basis of flowering regulation in mango, we isolated and characterized the MiSOC1, a putative mango orthologs for the Arabidopsis SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1/AGAMOUS-LIKE 20 (SOC1/AGL20) with homology-based cloning and RACE. The full-length cDNA (GenBank accession No.: KP404094) is 945 bp in length including a 74 bp long 5' UTR and a 189 bp long 3' UTR and the open reading frame was 733 bps, encoding 223 amino acids with molecular weight 25.6 kD. Both sequence alignment and phylogenetic analysis all indicated that deduced protein contained a conservative MADS-box and semi-conservative K domain and belonged to the SOC1/TM3 subfamily of the MADS-box family. Quantitative real-time PCR was performed to investigate the expression profiles of MiSOC1 gene in different tissues/organs including root, stem, leaves, flower bud, and flower. The result indicated MiSOC1 was widely expressed at different levels in both vegetative and reproductive tissues/organs with the highest expression level in the stems' leaves and inflorescences, low expression in roots and flowers. The expression of MiSOC1 in different flower developmental stages was different while same tissue -specific pattern among different varieties. In addition, MiSOC1 gene expression was affect by ethephon while high concentration ethephon inhibit the expression of MiSOC1. Overexpression of MiSOC1 resulted in early flowering in Arabidopsis. In conclusion, these results suggest that MiSOC1 may act as induce flower function in mango.

Keywords: MADS-box; MiSOC1; analysis; flowering; mango (Mangifera indica L).

FIGURE 1

Alignment of MiSOC1 predicted amino acid sequence with other SOC1 protein showing MIKC domains. Conserved domains (M, K) were boxed with black solid and red dashed line, respectively, while C-domain was located behind K-box. In C-domain, the SOC1 motif is highlighted with black dashed line box. The protein sequences of SOC1 genes aligned in this study were retrieved from NCBI. The GenBank accession numbers of 11 SOC1 genes were LcSOC1(AGS32267.1), CsSOC1(NP_001275772.1), BnSOC1(NP_001303107.1), CaSOC1(AHI85950.1), GhSOC1(AEA29618.1), PsSOC1(AGD88523.1), PaSOC1(ACO40488.1), GmSOC1(NP_001236377.1), CfSOC1(AGN29205.1), AtSOC1(NP_182090.1) and FvSOC1(AEO20231.1).

FIGURE 2

Phylogenetic analysis of MiSOC1 and its homologous sequences from various plant species. The proteins were initially aligned using Clustal W and were used for phylogenetic analysis using MEGA version 6.0 software. The phylogenetic tree was constructed using the neighbor-joining method. Bootstrap percentages were shown at dendrogram branch points. The accession numbers of the genes are PaSOC1(ACO40488.1), PmSOC1(AEO20229.1), PsSOC1(AGD88523.1), ScSOC1(AEO20234.1), RhSOC1(AEO20230.1), FvSOC1(AEO20231.1), CchSOC1(AHI85950.1), CcjSOC1(KYP64350.1), GmSOC1(NP001236377.1), GhSOC1(AEA29618.1), LcSOC1(AGS32267.1), CsSOC1(NP001275772.1), MiSOC1(AKJ85721.1), BnSOC1(NP001303107.1), BoSOC1(AGT96413.1), BrSOC1(NP001288813.1), BjSOC1(AFM77895.1), AlSOC1(AKX66088.1), CfSOC1(AGN29205.1), AtSOC1(NP182090.1), TaSOC1(BAF56968.1) and ZySOC1(AAG43199.1).

FIGURE 3

3D structural model of MADS-box domain of the MiSOCl protein.

FIGURE 4

Semi-quantitative RT-PCR (A) and Quantitative real-time RT-PCR (B) expression of MiSOC1 in different tissues of Mangifera indica. Carabao is shown. R: root; S: stem; L: leaves; Fb: flower buds; F: flowers. Total RNA was extracted from the indicated organs and RT-PCR was performed. Mango β-action was used as the endogenous control. The levels in roots were arbitrarily set to 1. Error bars represent the standard deviations of three technical PCR replicates.

FIGURE 5

Expression pattern of MiSOC1 at flowering stage of Mangifera indica. Carabao is shown. Fb: Flower bud, Bu: bud, Ef: early flower, Bl: blossom; Fl: Fruitlet. The relative expression ratio of each sample is compared to the control group which was blossom, respectively, and arbitrarily set to 1. Different letters represent significant differences at P < 0.05 according to Duncan’s multiple range tests. Error bars represent the standard deviations of three technical PCR replicates.

FIGURE 6

Expression pattern of MiSOC1 in different tissues of Mangifera indica. Kiett (A) and Jinhuang (B) is shown. R: root; S: stem; L: leaves; Fb: flower buds; F: flowers. Total RNA was extracted from the indicated organs and RT-PCR was performed. Mango β-action was used as the endogenous control. The levels in Flower bud were arbitrarily set to 1. Error bars represent the standard deviations of three technical PCR replicates.

FIGURE 7

Expression pattern of MiSOC1 in different tissues of Mangifera indica. Carabao under ethephon treatment is shown. R: root; S: stem; L: leaves; Fb: flower buds; F: flowers. Total RNA was extracted from the indicated organs and RT-PCR was performed. Mango action was used as the endogenous control. The levels in flower bud with water were arbitrarily set to 1. Different letters represent significant differences at P < 0.05 according to Duncan’s multiple range tests. Values are the average of three experiments with three biological replicates with three technical replicates.

FIGURE 8

Overexpression of MiSOC1 promotes flowering in Arabidopsis in Long-day condition and has fewer leaves. (A) The phenotype of MiSOC1 transgenic lines under Long-day condition. (B) Transgenic plants were confirmed by qRT-PCR with β-action as an internal reference. Values represent the means, and the error bars represent standard errors for three independent experiments. Different letters represent significant differences at P < 0.05 according to Duncan’s multiple range tests. (C) The number of rosette leaves during flowering in transgenic plants and wild-type plants.