2.1 Basic physical and chemical properties
Finally, we identified 10 SRS genes in quinoa, which were named SRS01-SRS10. The coding sequence (CDSs) of the members of this family is between 432-1113 nucleotides, and the coding amino acid sequence is between 143-370 amino acids (Table 1), with an average of 244 amino acids. Except for CqSRS01 and CqSRS02, the PI of the coding proteins is less than 7, and the hydrophobicity index of all CqSRS proteins is less than 0, indicating that these proteins are hydrophilic. Subcellular localization prediction showed that most CqSRS genes were located in the cytoplasm (5 CqSRS genes), and a few were located in the nucleus, plasma membrane and mitochondria. The structure and stability of CqSRS proteins are determined by the instability index, which provides an estimate of protein stability. In this study, 6 CqSRS proteins were unstable, with the instability index greater than 40. 4 CqSRS proteins may be stable, with an index between 32.45 and 39.45.11.
2.2 Evolutionary Relationships and Classification of SRS Genes
To study phylogenetic relationships between the SRS proteins of quinoa, we constructed phylogenetic trees from 44 protein sequences of Arabidopsis (11), maize (9), tomato (9), spinach (5) and Quinoa (10). According to the topological structure of the tree, all plants share a common ancestor with the SRS genes. Meanwhile, according to their homology, they are divided into 3 subfamilies (Fig 1 and S1 Table). The first group has 11 SRS genes, the second group contains 10 genes, and the third group is the largest, containing 23 genes. At the same time, we can observe that there are 14 pairs of orthologous genes in these 5 species (6 pairs in quinoa, CqSRS06/SlSRS07, CqSRS04/SlSRS03, CqSRS05/AtSRS06, CqSRS02/AtSRS01, CqSRS08/ZmSRS05), and 5 pairs of paracentric homologous gene pairs (2 pairs in quinoa: CqSRS03/CqSRS09, CqSRS01/CqSRS10). There are 2 pairs of homologous genes between quinoa and Arabidopsis, indicating that there is no obvious difference between these two species in the evolutionary process.
2.3 Chromosomal location and gene duplication analysis
To verify the relationship between genetic differentiation and gene replication, we identified the chromosomal locations of CqSRS genes (Fig 2). In this study, the chromosomal locations of CqSRS gene family members were obtained through the quinoa genome (David et al. 2017). Ultimately, 10 CqSRS genes were located on the 9 chromosomes of quinoa (quinoa has a total of 18 chromosomes), with 1 SRS gene on 8 chromosomes except chromosome 9, which contains 2 SRS genes.
The replication of CqSRS genes was further tested. Previous studies showed that 5 or fewer genes located within the range of 100kb on the same chromosome are usually considered as tandem repeats (McGregor et al. 2017), so there are no tandem repeats in this study. We based on two conditions (comparison rate of two genes > 75%, comparing similarity > 75%), screening for repeat genes, identified to 4 of duplicated genes (Table 2), and they respectively located on different chromosomes and thus belongs to the duplicated gene fragments, and repeated occur between 6.830-14.151 MYA. The history of the selection acting on the coding sequence can be measured in terms of the ratio of non-synonymous substitutions to synonymous substitutions (Ka/Ks). Ka/Ks<1 was selected for purification. When the two sequences drift in neutral and special, Ka/Ks=1. At specific sites of positive selection, Ka/Ks>1. Ka/Ks values of 4 gene pairs in this study were all less than 1, indicating that the evolution of all gene pairs was mainly influenced by purification selection, and purification selection could inhibit the differentiation of duplicate genes.
2.4 Analysis of gene structure and conserved motifs
On the one hand, the diversity of gene structure reflects the evolutionary relationship of gene families. Meanwhile, the intron-exon pattern plays a key role in gene function. Therefore, we analyzed the exon/intron pattern of members of this family by comparing the coding sequence with the corresponding genomic DNA sequence. Results showed that the number of exon CqSRS between 2 and 5, and at the same subfamily gene has a similar introns/exon mode. For example, the number and length of exons of corresponding genes in subfamilies 1, 2 and 3 are highly similar, and the genes are highly homologous to each other, suggests that they are in the process of evolution is derived from a common ancestor, or maybe the result of a genetic replication (Fig 3). The conservative motifs of CqSRS proteins were analyzed by using MEME and 10 conserved motifs were selected. It was found that Motif 4 exists in all CqSRS genes, Motif 1 exists in most CqSRS genes, and Motif 3, 5, 6 and 8 only exist in CqSRS03 and CqSRS09. Motif 9 may be the basis for the division of CqSRS01 and CqSRS02 in the same branch. Most CqSRS genes with similar gene structure have the same motif compositions and similar functions.
2.5 Cis-acting element analysis and construction of protein interaction network
In order to study the cis-acting elements in the CqSRS genes promoter region, we analyzed the promoter sequence of CqSRS genes (2000bp upstream of translation starting point) by PlantCARE. We found that all CqSRS genes promoter region contained one or more TATA-box. At the same time, we found a total of 44 elements related to plant hormone response elements, light response elements, stress response elements and tissue-specific expression in the upstream region of the promoter (Fig 4 and S3 Table). The light response element was the most cis-acting element, followed by plant hormone and stress response element, and the tissue-specific expression element was the least. Plant hormones such as auxin, abscisic acid, gibberellin and jasmonic acid play a key role in plant resistance to adversity. In this study, CqSRS genes contained a variety of hormone-related elements. ABRE, CGTCA-motif, TGACG-motif and other plant hormone elements existed in all CqSRS genes in the form of a single copy or multiple copies. Some genes (CqSRS02, CqSRS05, CqSRS07, CqSRS06 CqSRS07, CqSRS08 and CqSRS10) contained 5 hormone response elements, including abscisic acid (ABRE), AuxRE (AuxRE, AUXRR-core, CGTCA-motif and TGA-Box), salicylic acid (TCA-element), gibberellin (GARE, P-box and TATC-Box) and methyl jasmonate (TGACG-motif). CqSRS genes also contain some tissue-specific elements, including meristem expression elements (CAT-box) and endosperm expression elements (GCN4_motif and AACA-motif). In addition, the family also contain a small number of stress response elements, including low-temperature response elements (LTR), drought induction elements (MBS), and defense and stress response elements (TC-rich repeats).
To further investigate which protein interact with SRS family members, a network of protein interaction between quinoa and Arabidopsis was predicted using the STRING database. As can be seen from the figure below, 10 CqSRS proteins appear in the known Arabidopsis protein interaction network (Fig 5). Among them, the protein sequence of AtSTY1 is highly similar to that of CqSRS07, AtSTY1 gene, as a transcriptional activator, can bind to the DNA on 5' -ACTCTAC 3' and promote the expression of auxin homeostasis regulation genes (such as YUC gene), as well as genes affecting stamen development, cell amplification and flowering time, so CqSRS07 gene may have a similar function. AtLRP1 gene has been identified as an auxin-induced gene, and its expression is regulated by histone deacetylation, so the expression of CqSRS01 and CqSRS02 may also be regulated by auxin signal (Singh et al. 2020). 5 CqSRS genes (CqSRS04, CqSRS05, CqSRS06, CqSRS08 and CqSRS10) are similar to AtSHI gene, revealing their synergistic effect with other related proteins (NGA3 and YUC1) to regulate pistillate, stamen and leaf development in a dose-dependent manner and control apical basal configuration, and promote pistil development and stigma formation, and affect the development of blood vessels during pistil development.
2.6 Secondary structure analysis and tertiary model prediction
In order to better understand the structural characteristics of CqSRS proteins, a third-level model of the protein family was predicted using Swiss-model, and the results showed that members in the same subgroup had similar third-level structures (Fig 6). The secondary structure consists of random coil（Cc）, extended strand（Ee）, and alpha helix（Hh）, of which random coil account for the largest proportion (more than 50%)(S4 Table).
2.7 RNA-seq analysis
We used transcriptome data to study the expression patterns of genes in this family. The results of heatmap showed that most CqSRS genes showed a low expression under different treatments (Fig 7 and S5 Table). For example, CqSRS04, CqSRS05, CqSRS06 and CqSRS10. CqSRS01, CqSRS02 and CqSRS03 genes are highly expressed in roots under high temperature, low phosphorus, drought and salt stress, and these genes may play a key role under abiotic stress. In addition, the expression of CqSRS genes in tissues and organs at different development stages of quinoa was also significantly different. Almost all the genes high expression in Apical meristems and Flowers of white sweet quinoa. Most genes (except CqSRS08) are low expression in leaves. The expression pattern of CqSRS08 was different from that of other proteins. The expression of CqSRS08 was high in all tissues, especially in leaves up to 43, indicating that some SRS genes have the characteristics of tissue expression.
2.8 Expression profiling of CqSRS genes in different treatments
Stress seriously affects the growth and development of plants, so qRT-PCR was used to analyse the expression patterns of the family members in roots under stress (Fig 8 and S6-S7 Tables). The results showed that all SRS family genes were responsive to SA, NaCl and low-temperature treatments. The expression levels of different CqSRS genes were significantly different under different stress. In SA treatment, some genes (CqSRS02, CqSRS03, CqSRS05 and CqSRS06) showed the same pattern of first increasing and then decreasing, and some genes (CqSRS01, CqSRS04, CqSRS07, CqSRS08, CqSRS09 and CqSRS010) showed the lowest expression after 8 h treatment. Under NaCl and low-temperature treatment, most of the genes had the same expression pattern (2 h or 12 h expression level was extremely significant), and the expression level of the treatment was significantly higher than that of the control group. However, the expression of CqSRS10 gene in NaCl and low temperature was lower than that in control. These results showed that the CqSRS gene family members in most roots were strongly induced by 200 mmol/L NaCl, 200 mml/L SA and 4℃ treatment under different treatments, and only a few members were not sensitive to abiotic treatment.