The SBP-box protein family is a type of plant-specific transcription factor which regulates the expression of downstream genes by combining with cis-acting elements in the promoter region of downstream genes. The number of members of this family varies greatly among different species, and 49 SBP genes have been identified in soybean (Schmutz et al.2010). This study identified 23 quinoa SBP genes. This number variation makes the function of SBP transcription factors more diversified. More SBP genes are in the soybean genome, indicating that the soybean SBP gene family has experienced more complex amplification, loss, and evolutionary processes. The 23 CqSBP genes are distributed on 12 chromosomes. The number of amino acids, isoelectric points, and molecular weights of CqSBP proteins is quite different. This may be due to the family members' different functions during growth and development, amino acid composition, and protein structure. The difference may lead to differences in the function of SBP gene family members. Interestingly, we found that all CqSBP genes are located in the nucleus, indicating that CqSBP can be a transcription factor in the nucleus.
Gene duplication is of great significance to the evolution of gene families, mainly because gene duplication can provide the most primitive material for generating new genes, and the generation of new genes promotes new functions (Rensing., 2014). There are three main ways of plant gene replication: fragment replication, tandem replication, transposition events such as retrotransposition, and repeated transcription. Fragment duplication is the most important way because most plants undergo a chromosome doubling process and retain many repeated chromosome fragments in the genome (Wang et al. 2012). This study identified that 11 pairs of homologous genes were generated by fragment duplication, and 1 pair of homologous genes was amplified by tandem duplication, indicating that the amplification of the quinoa gene family was mainly amplified by fragment duplication. SPL10/SPL11 in Arabidopsis has the same gene structure, the sequence is highly similar (82.1%), and they are adjacent to each other on the chromosome. The tandem repeat mechanism may produce them. The corresponding SBP-box in Quinoa has tandem replication. The family members CqSBP06/CqSBP07 also have high homology. Quinoa is a heterotetraploid plant, and its genome has undergone a process of doubling during evolution, so many genes exist in multiple copies in the quinoa genome. Recent studies (Jarvis et al.2017) have shown that a whole-genome duplication event occurred in Quinoa between 3.3–6.3 Mya (million years ago), supporting the idea that genes exist in multiple copies. This study found that multiple copies of SBP genes are scattered on different chromosomes. Microcollinearity analysis showed that there are 8 pairs of SBP paralogous genes in the quinoa genome, which provides favorable evidence for the doubling of the quinoa genome.
From a phylogenetic point of view, SBP genes are composed of multiple members in most plants. For example, 3, 4, and 8 SBP homologous gene pairs were identified in Arabidopsis, rice, and soybean genomes. They belong to the horizontal homologous genes, are in the same evolutionary branch, and are formed after the occurrence of species. Compared with Arabidopsis and rice, Quinoa has more SBP homologous gene pairs, which fully indicates that more repetitive events of SBP genes occur after quinoa speciation. Generally speaking, SBP genes with complete SBP functional domains can often find EST sequences, which means that these SBP genes have transcriptional activity. The 23 CqSBP genes in this study all included typical SBP functional domains.
The C-terminus of the conserved domain of the SBP transcription factor is the nuclear localization signal region. When analyzing the conserved domains of SBP, this study found that the domain of quinoa SBP protein contains about 79 amino acid residues and has two zinc finger structures, which are C3H (C-C-C-H) C2HC (C-C-H-C) types. In addition, most transcription factors have an NLS site at the C-terminus of the SBP conserved domain. In addition, CqSBP genes located in the same branch share similar intron/exon structures. And most CqSBPs of the same branch have similar motifs. Therefore, genes in the same phylogenetic group may have similar roles in Quinoa. This indicates that the evolution of the SBP-box gene family may be closely related to the diversity of gene structure. In addition to the conserved CqSBP motif, several unique group-specific motifs have also been observed, such as CqSBP05, CqSBP06, CqSBP07, and CqSBP13 subfamily The motifs in 2, 5, 6, 7, 8, 9 and 10. These specific motifs may be important for the specific role of the CqSBP genes, and their functional differentiation may have appeared during the evolution of different lineages.
Gene expression profile analysis showed that the expression patterns of genes in this family are different in different tissues. These results may be an important research resource for further revealing the function of CqSBP genes in quinoa development. Most SBP genes are widely expressed in the meristems, flowers, inflorescences, petioles, internodes, stems, and leaves of Quinoa, which indicates their key role in these biological processes. At the same time, most CqSBP genes are highly expressed in the apical meristem, which indicates that they are widely involved in cell differentiation. Studies have shown that AtSPL8 mediates anther development, flowering, cell differentiation, floral organ development, and stamen filament elongation (Zhang et al. 2007; Unte et al. 2003). BrcSPL8 plays an important role in developing Chinese cabbage flowers (Zhang et al.2017). In this study, CqSBP01 and CqSBP02, homologous to AtSPL8, are highly expressed in inflorescence and apical meristems. It is inferred that they may be involved in distinguishing meristems and flower formation. CqSBP08 and CqSBP10 are highly expressed in stems, and they may participate in stem development and regulation of organ formation, which has been verified in the study of the homologous gene AtSPL9 (Zhang et al.2020).
AtSPL14 plays a role in developing normal plant structures and is sensitive to fumonisin B1 (Stone et al.2005). It is highly expressed in all organizations. OsSPL6 and OsSPL15 are also highly expressed in all tissues. In our study, CqSBP16 (the homologous gene of AtSPL14) is highly expressed in all tissues, and it may affect the plant structure of Quinoa. The qualitative analysis of the role of the quinoa SBP transcription factor in the growth and development of Quinoa is of great significance to the study of the function of the Quinoa SBP gene. It provides a certain theoretical basis for studying the regulation mechanism of cotton fiber (with economic value). Therefore, increasing the research on these SBP genes may help better understand specific physiological processes and subsequent agricultural genetics research. Q-TR-PCR showed that CqSBP01, CqSBP02, and CqSBP13 expressed extremely significantly under salt stress, indicating that these three genes may play a key role in salt stress. Wang (Wang et al.2009) analyzed the regulatory network related to SPL genes and found that 112 genes are closely related to SPL genes, and the promoters of these genes all contain a core motif of GATC, speculating that SBP transcription factors may be involved in plant tissues development, biotic and abiotic stress response, and activation of other transcription factors and membrane proteins. At the same time, the SPL gene may also be involved in the metabolism of glucose, inorganic salts, ATP, and the transport of carbohydrates. The SBP gene was discovered in the gene regulation network that studies the path of flower formation, so it is considered to be closely related to flower development. In recent years, it has been discovered that the SBP gene has a wide range of biological functions. The SBP gene has been found in many plants, but its function is still poorly understood. One of the reasons is that almost all functional studies are carried out through mutants, for example, by constructing over-expression or silencing vectors of related genes, then using transgenic technology to transfer them into corresponding plants, and finally by observing the phenotype of the transgenic plants. Explain the function of genes.