3.1 Identification of SDR genes in B. napus genome
A total of 142 BnaSDR genes were identified from B. napus genome by blast with 55 AtSDR sequences. The results of chromosome location shown that there were 141 BnaSDR genes anchored in 19 B. napus chromosomes (Fig. 1). However, Bnascaffold2694G0000200ZS had not been anchored to any chromosome. We also found that 141 BnaSDR genes were unevenly distributed on the A and C sub-genomes. There were 67 and 74 BnaSDR genes were located on A and C sub-genomes, respectively. Among them, chromosome C04 contained the maximum number of BnaSDRs (17), and chromosome A06 and C06 had only two BnaSDRs. Furthermore, BnaSDR gene clusters, contain more than five BnaSDRs in a 20 M region, were identified on chromosomes A04, A05, C03 and C04 (Fig. 1).
3.2 Evolution of SDR family in B. napus
To understand the evolutionary relationships and functions among the predicted BnaSDR proteins, a phylogenetic tree was created with 142 BnaSDR protein sequences and 55 AtSDR protein sequences by MEGA7.0. Phylogenetic tree results shown that 142 BnaSDR proteins were divided into four distinguishing groups (Group A–D; Fig. 2). The subgroup A contained the least number of SDR members, only 4 AtSDRs and 9 BnaSDRs. But Subgroup D, a largest group, contains 20 AtSDRs and 56 BnaSDRs. Subgroup B contained 11 AtSDRs and 27 BnaSDRs, subgroup C contained 20 AtSDRs and 50 BnaSDRs.
3.3 physicochemical properties of BnaSDR proteins
In order to clarify the basic characteristics of SDR family proteins in B. napus, the physicochemical properties of 142 BnaSDR proteins were analyzed. Results shown that the sequence length of BnaSDR proteins were ranged from 92 to 501 aa, with an average length of 306 aa. The largest relative molecular mass is BnaA04T0077900ZS (55188.59 Da), the smallest relative molecular mass is BnaC05T0510200ZS (9801.25 Da), and the average relative molecular mass is 33170.24 Da. The average theoretical pI is 7.37, and most BnaSDR proteins are alkaline. The instability index of BnaSDR proteins were between 10.35 and 66.84, and only 13.38% of the proteins were unstable proteins (Table S1). The average hydrophobicity index of BnaSDR proteins was between − 0.603 and 0.381, and 61.27% of the protein showed hydrophobicity (Table S2; Fig. 3). Results of subcellular localization showed that most of the BnaSDR proteins (71, 50%) were located in chloroplasts, 33.10% BnaSDR proteins located in cytoplasm, and some BnaSDRs were located in peroxisomes (3.52%), plasma membrane (3.52%), nucleus (2.82%), endoplasmic reticulum (2.82%), cytoskeleton (2.11%) and extracellular matrix (1.41%). Only one BnaSDR protein (BnaC04T0186800ZS), homologous to At2g29330, was located on the Golgi apparatus (Table S1).
By comparing the physicochemical properties of different subgroups (Fig. 3), it was found that the number of amino acids, molecular weight and instability index of subgroup B were the highest among four subgroups. And the unstable proteins were mainly distributed in subgroup B. The average theoretical pI of subgroup A and subgroup C was greater than 7, most of the BnaSDRs was alkaline. most of the BnaSDRs in subgroup B was neutral. BnaSDRs in subgroup C was mostly acidic. The grand average of hydrophobicity of subgroup A was the highest; in addition, the proteins of A, C and D subgroups were almost hydrophobic proteins, and the B subgroup was a hydrophilic protein.
3.4 conserved motifs of BnaSDR proteins
To further understand the protein sequence features of BnaSDRs, the conserved motifs of each protein were identified using the MEME online software. we found that almost all of BnaSDR proteins contained motif 4 (95.77%), indicating that motif 4 is relatively conserved in the SDR family. However, only 14.79% SDR proteins contained the motif 12 (Fig. 4A, Figure S1).
Results of the distribution of conserved motifs in different proteins has shown that proteins in the same group have similar motif distribution. It was found that motif 1, motif 2, motif 5 and motif 12 only existed in the members of subgroups B, C and D. In addition, the proteins containing motif 1, motif 2 and motif 5 are mainly in subgroup D. BnaSDRs containing motif 9, motif 10 and motif 11 are mainly in subgroup C (Fig. 4B). These indicating that the proteins in different subgroups may have large functional differentiation.
3.5 gene structure and cis-acting elements of BnaSDR genes
To character structures of BnaSDR genes, gene length, CDS length, intron number, and cis-acting elements were investigated. By aligning CDS and genomic sequences, we found that the structure and intron number were quite different among BnaSDR genes, but roughly similar among a subgroup. there were 2 to 12 introns of BnaSDR genes. However, the number and location of BnaA09T0182700ZS introns are very different from other branches in the same subgroup (Figure S2). These indicating that functional differentiation of BnaA09T0182700ZS may occur.
To explore cis-acting elements in the promoter region of BnaSDR genes, the online promoter database Plant Care was employed in present study. Results shown that there were 1558 (14 no duplication) cis-acting elements in the BnaSDR promoter regions. There were 856 hormone response regulatory elements (5 no duplication), including auxin-responsive element, gibberellin-responsive element, abscisic acid responsiveness, salicylic acid responsiveness and CGTCA-motif responsive element. We also found that there were 42.99% and 36.10% BnaSDR genes containing CGTCA-motif response element and abscisic acid response element, respectively (Table 2, Figure S3). These indicated that the expression of the gene might be regulated by hormone signaling substances.
Besides, there were 625 (5 no duplication) environmental stress responses elements, including light responsive element, Anaerobic induction, Defense and stress responsiveness, Low-temperature responsiveness, MYB binding site involved in drought-inducibility. And the remaining 77 cis-elements (4 no duplication) are related to tissue expression, including Meristem expression, MYB binding site involved in flavonoid biosynthetic genes regulation, endosperm expression and phytochrome down-regulation expression (Table 2, Figure S3). These results indicated that the functional diversity of BnaSDR genes has been developed during the growth and development of B. napus.
Table 2
Distribution cis-acting elements of different subgroup BnaSDR genes
Groups | Group A | Group B | Group C | Group D | Total |
Phytohormones responsive | Auxin | 5 | 16 | 13 | 20 | 54 |
GA | 4 | 6 | 24 | 26 | 60 |
ABA | 13 | 61 | 121 | 114 | 309 |
SA | 3 | 13 | 26 | 23 | 65 |
MeJA | 16 | 66 | 124 | 162 | 368 |
Environmental responsive | Light | 15 | 29 | 63 | 59 | 166 |
Anaerobic | 14 | 43 | 74 | 120 | 251 |
Defense/stress | 10 | 12 | 16 | 22 | 60 |
Low-T | 6 | 16 | 14 | 36 | 72 |
Drought | 6 | 8 | 20 | 42 | 76 |
Other responsive | Meristem | 1 | 8 | 17 | 26 | 52 |
Endosperm | 1 | 1 | 6 | 6 | 14 |
Phytochrome | 1 | 1 | 0 | 0 | 2 |
Flavonoid | 1 | 1 | 2 | 5 | 9 |
3.6 Tissue expression profiles of BnaSDR genes
To explore tissue-specific expression profiles of the BnaSDR genes, 37 tissues (including 11 tissues and 26 seeds at the different development stages) were selected from the database (https://biodb.swu.edu.cn/brassica/). The results showed that the 142 BnaSDR genes widely expressed in different tissues (Figure S4). But the expression pattern of BnaSDR genes in different groups were largely different. The expression level of genes in subgroup A in buds and filaments were higher than those in other groups. However, the expression level of genes in subgroup A were lower in siliques compared with genes in other groups. BnaSDR genes in subgroup D was highly expressed in stems, leaves, sepals, cotyledons and siliques, but not in pollen. In subgroup B, BnaA02T0152200ZS, BnaC02T0194900ZS, BnaA03T0253100ZS and BnaC03T0300500ZS were only highly expressed in seeds.
The expression level of BnaSDR genes in seeds from 14 to 64 days after flowering were also compared between different subgroups (Fig. 5). As a whole, the expression levels of BnaSDR genes in subgroup A and D were gradually decreased with the development of seeds. However, gene expression level of BnaSDR genes were greatly fluctuated in subgroup B. Especially, the expression level of BnaA02T0152200ZS, BnaA03T0253100ZS, BnaC03T0300500ZS and BnaC03T0290800ZS were significantly increased at the late stage of seed development (64 days after flowering). In subgroup C, gene expression levels of most BnaSDR genes were undulated with the development of seeds, except to BnaA01T0256500ZS, BnaC01T0313900ZS and BnaA05T0476100ZS, in which the expression levels were rapidly raised at the late stage of seed development (64 days after flowering).
3.7 BnaSDR genes expression in dormant seed
Based on the phylogenetic and gene expression data, seven representative BnaSDR genes were selected from the subgroups B and C for further study. Expression level of these seven genes in weak dormancy (T) or nondormancy (S) seeds were analyzed by RT-PCR (Fig. 6A). Results shown that the expression levels of four BnaSDRs (BnaC03T0290800ZS, BnaA01T0256500ZS, BnaC01T0313900ZS, BnaA05T0476100ZS) in fresh seeds of T line were significantly higher than those in S line. While the expression levels of the other three genes were no significantly different between two lines (Fig. 6B). These indicating that BnaC03T0290800ZS, BnaA01T0256500ZS, BnaC01T0313900ZS, and BnaA05T0476100ZS might relate to seed dormancy.
Comparing the gene expression levels before and after seed germination, it was found that seed germination treatment significantly reduced the expression levels of 7 genes in both T and S lines. We also found that the expression levels in germinated seeds after 24 hours were lower than those in non-germinated seeds (Fig. 6B). These indicating that the gene expression levels gradually decreased with the process of seed germination.