Identification of the DMP proteins in oat
Thirty-three DMP sequences were identified and renamed as AsDMP1 to AsDMP33 according to their positions on the chromosome (Table 1). In addition, the physicochemical properties of these oat DMP genes were determined, including amino acid sequence length (aa), isoelectric point(PI), protein molecular weight (kDa), instability index, fat index, and subcellular localisation. All 33 DMP genes encode amino acid lengths ranging from 169 (AsDMP6) to 325 (AsDMP24) aa; Theoretical equivalence points range from 5.26 (AsDMP28) to 9.04 (AsDMP33); Molecular weight range 17.88(AsDMP6) to 35.50(AsDMP24) kDa; The predicted fat index range was 76.04 to 107.62. Fat index indicates thermal stability of proteins. Hydrophilicity and hydrophobicity of proteins is one of the most important factors affecting the structural stability of proteins. All AsDMP proteins were analysed and found to be hydrophilic (Fig. 1), indicating their transmembrane nature. In addition, all AsDMP proteins had transmembrane structural domains ranging from 3 to 4 in number, and none of the AsDMP proteins possessed a signal peptide, suggesting that all AsDMP proteins are transmembrane unsecreted proteins. Predictions of the subcellular localisation of DMP proteins indicate that all proteins are localised to the plasma membrane, which is consistent with the function of DMP. In addition, AsDMP14, AsDMP19, AsDMP24, AsDMP25, AsDMP26 and AsDMP32 were localised extracellularly, as well as AsDMP15 and AsDMP21 in chloroplasts, and these results suggest that the DMPs also regulate biological processes inside and outside the plasma membrane.
Table 1
The characterization of oat DMP genes in this study
Gene name | Gene ID | Protein length (aa) | Isoeletric point (PI) | Molecular Weight (MW)/kDa | Instability index | Aliphatic index | Transmembrane domains | signal peptide | Predicted subcellular localization |
AsDMP1 | AVESA.00010b.r2.1AG0036510.1 | 239 | 6.50 | 25.35 | 50.20 | 96.78 | 4 | NO | PlasmaMembrane |
AsDMP2 | AVESA.00010b.r2.1AG0004840.1 | 236 | 6.03 | 25.01 | 44.79 | 97.58 | 4 | NO | PlasmaMembrane |
AsDMP3 | AVESA.00010b.r2.1DG0155630.1 | 235 | 6.28 | 24.95 | 47.51 | 99.23 | 4 | NO | PlasmaMembrane |
AsDMP4 | AVESA.00010b.r2.2CG0263910.1 | 284 | 6.22 | 30.45 | 34.11 | 97.29 | 4 | NO | PlasmaMembrane |
AsDMP5 | AVESA.00010b.r2.2DG0399840.1 | 215 | 8.40 | 22.92 | 37.84 | 90.37 | 4 | NO | PlasmaMembrane |
AsDMP6 | AVESA.00010b.r2.2DG0382070.1 | 169 | 7.58 | 17.88 | 47.24 | 86.75 | 3 | NO | PlasmaMembrane |
AsDMP7 | AVESA.00010b.r2.3AG0415880.1 | 216 | 6.03 | 22.95 | 31.51 | 90.37 | 4 | NO | PlasmaMembrane |
AsDMP8 | AVESA.00010b.r2.3CG0462960.1 | 216 | 6.17 | 23.21 | 28.56 | 95.32 | 4 | NO | PlasmaMembrane |
AsDMP9 | AVESA.00010b.r2.3CG0495930.1 | 173 | 7.73 | 18.60 | 25.81 | 103.12 | 4 | NO | PlasmaMembrane |
AsDMP10 | AVESA.00010b.r2.3CG0514780.1 | 219 | 7.69 | 24.54 | 50.08 | 98.36 | 4 | NO | PlasmaMembrane |
AsDMP11 | AVESA.00010b.r2.3CG0515720.1 | 231 | 6.82 | 24.79 | 43.59 | 83.25 | 4 | NO | PlasmaMembrane |
AsDMP12 | AVESA.00010b.r2.3CG0515740.1 | 237 | 7.69 | 25.30 | 32.07 | 83.21 | 4 | NO | PlasmaMembrane |
AsDMP13 | AVESA.00010b.r2.3DG0517300.1 | 216 | 6.03 | 22.93 | 30.89 | 92.18 | 4 | NO | PlasmaMembrane |
AsDMP14 | AVESA.00010b.r2.3DG0543000.1 | 226 | 5.92 | 24.87 | 42.77 | 96.73 | 3 | NO | PlasmaMembrane、Extracellular |
AsDMP15 | AVESA.00010b.r2.4AG0601280.1 | 230 | 9.01 | 24.65 | 42.54 | 76.04 | 4 | NO | PlasmaMembrane、 Chloroplast |
AsDMP16 | AVESA.00010b.r2.4AG0640750.1 | 218 | 8.30 | 24.35 | 44.69 | 99.72 | 4 | NO | PlasmaMembrane |
AsDMP17 | AVESA.00010b.r2.4AG0641780.1 | 233 | 6.29 | 24.95 | 46.68 | 83.82 | 4 | NO | PlasmaMembrane |
AsDMP18 | AVESA.00010b.r2.4CG1263690.1 | 185 | 6.70 | 19.46 | 22.93 | 107.62 | 4 | NO | PlasmaMembrane |
AsDMP19 | AVESA.00010b.r2.4CG1314570.1 | 193 | 8.38 | 20.80 | 32.02 | 82.44 | 4 | NO | PlasmaMembrane、Extracellular |
AsDMP20 | AVESA.00010b.r2.4DG0745150.1 | 181 | 8.46 | 20.13 | 39.60 | 84.09 | 4 | NO | PlasmaMembrane |
AsDMP21 | AVESA.00010b.r2.4DG0745170.1 | 229 | 7.75 | 24.56 | 44.47 | 76.42 | 4 | NO | PlasmaMembrane、 Chloroplast |
AsDMP22 | AVESA.00010b.r2.4DG0768120.1 | 177 | 6.41 | 19.36 | 42.26 | 86.05 | 3 | NO | PlasmaMembrane |
AsDMP23 | AVESA.00010b.r2.4DG0769210.1 | 233 | 6.07 | 24.97 | 46.92 | 81.33 | 4 | NO | PlasmaMembrane |
AsDMP24 | AVESA.00010b.r2.4DG0769220.1 | 325 | 8.27 | 35.50 | 30.29 | 77.78 | 4 | NO | PlasmaMembrane、Extracellular |
AsDMP25 | AVESA.00010b.r2.5AG0809890.1 | 193 | 8.39 | 20.78 | 34.74 | 84.46 | 4 | NO | PlasmaMembrane、Extracellular |
AsDMP26 | AVESA.00010b.r2.5DG0998080.1 | 193 | 8.39 | 20.78 | 34.74 | 84.46 | 4 | NO | PlasmaMembrane、Extracellular |
AsDMP27 | AVESA.00010b.r2.6AG1010130.1 | 210 | 7.60 | 21.65 | 39.73 | 82.90 | 4 | NO | PlasmaMembrane |
AsDMP28 | AVESA.00010b.r2.6AG1060270.1 | 212 | 5.26 | 21.92 | 25.48 | 97.22 | 4 | NO | PlasmaMembrane |
AsDMP29 | AVESA.00010b.r2.6AG1060280.1 | 185 | 8.38 | 19.38 | 23.78 | 106.05 | 4 | NO | PlasmaMembrane |
AsDMP30 | AVESA.00010b.r2.6CG1110230.1 | 243 | 8.85 | 26.01 | 39.66 | 80.78 | 4 | NO | PlasmaMembrane |
AsDMP31 | AVESA.00010b.r2.7AG1213710.1 | 214 | 8.78 | 22.84 | 36.62 | 92.62 | 4 | NO | PlasmaMembrane |
AsDMP32 | AVESA.00010b.r2.7CG0697010.1 | 227 | 6.37 | 24.56 | 43.24 | 76.96 | 4 | NO | PlasmaMembrane、Extracellular |
AsDMP33 | AVESA.00010b.r2.7DG1394100.1 | 213 | 9.04 | 22.80 | 40.14 | 90.75 | 4 | NO | PlasmaMembrane |
Protein structure prediction of oat DMP family members
The main protein secondary structures are α-helix, β-folding, β-turning, irregularly coiled and extended chains. The predicted secondary structure based on the amino acid sequence of oat AsDMP was found to consist mainly of α-helices, β-turns, irregularly coiled and extended chains (Table 2). In addition, α-helix and random curl account for a large proportion in the secondary structure of all AsDMPs members, and it can be determined that the amino acid secondary structure of AsDMPs is mainly composed of α-spiral and random coil. By predicting the tertiary structures of oat DMP family members' proteins, the tertiary structures of AsDMP1, AsDMP2 and AsDMP3 proteins, the tertiary structures of AsDMP4, AsDMP18, AsDMP20, AsDMP28 and AsDMP29 proteins, the tertiary structures of AsDMP5, AsDMP7, AsDMP8, AsDMP13 and AsDMP31 proteins, the tertiary structures of AsDMP10, AsDMP16, and AsDMP22 proteins, the tertiary structures of AsDMP11, AsDMP12, AsDMP15, AsDMP21, AsDMP23, and AsDMP24 proteins, the AsDMP19, AsDMP25, and AsDMP26 tertiary structures of AsDMP19, AsDMP25 and AsDMP26 proteins, and the tertiary structures of AsDMP27 and AsDMP30 proteins were similar to a high degree, presumably with some functional similarity, but showed different morphologies from each other and differed from the tertiary structures of the rest of the family members, suggesting a diversity of tertiary structures of the AsDMP family members (Fig. 2).
Table 2
Prediction of secondary structure of DMP gene family proteins in oats(%)
name
|
Alpha-helix
|
Extended strand
|
Random coil
|
Beta turn
|
AsDMP1
|
41.84
|
11.72
|
40.17
|
6.28
|
AsDMP2
|
41.53
|
11.86
|
40.68
|
5.93
|
AsDMP3
|
40.43
|
11.91
|
41.70
|
5.96
|
AsDMP4
|
46.83
|
13.32
|
35.92
|
4.93
|
AsDMP5
|
39.53
|
15.35
|
38.60
|
6.51
|
AsDMP6
|
45.56
|
15.38
|
32.54
|
6.51
|
AsDMP7
|
40.28
|
13.43
|
41.67
|
4.63
|
AsDMP8
|
41.20
|
12.04
|
41.67
|
5.09
|
AsDMP9
|
44.51
|
16.18
|
32.37
|
6.94
|
AsDMP10
|
42.01
|
17.35
|
36.99
|
3.65
|
AsDMP11
|
35.93
|
16.88
|
42.42
|
4.76
|
AsDMP12
|
34.18
|
18.99
|
41.77
|
5.06
|
AsDMP13
|
36.57
|
16.20
|
43.06
|
4.17
|
AsDMP14
|
45.13
|
11.06
|
37.17
|
6.64
|
AsDMP15
|
33.91
|
15.22
|
45.22
|
5.65
|
AsDMP16
|
39.45
|
18.81
|
35.78
|
5.96
|
AsDMP17
|
39.48
|
15.02
|
40.34
|
5.15
|
AsDMP18
|
43.78
|
14.59
|
34.59
|
7.03
|
AsDMP19
|
37.82
|
16.06
|
41.45
|
4.66
|
AsDMP20
|
44.20
|
14.92
|
34.25
|
6.63
|
AsDMP21
|
37.12
|
14.85
|
41.05
|
6.99
|
AsDMP22
|
31.64
|
21.47
|
40.68
|
6.21
|
AsDMP23
|
40.77
|
16.31
|
37.34
|
5.58
|
AsDMP24
|
41.54
|
14.15
|
39.38
|
4.92
|
AsDMP25
|
39.90
|
13.99
|
38.86
|
7.25
|
AsDMP26
|
39.90
|
13.99
|
38.86
|
7.25
|
AsDMP27
|
35.71
|
14.76
|
44.29
|
5.24
|
AsDMP28
|
41.98
|
13.21
|
38.68
|
6.13
|
AsDMP29
|
47.03
|
12.97
|
34.05
|
5.95
|
AsDMP30
|
39.51
|
12.76
|
42.39
|
5.35
|
AsDMP31
|
36.45
|
18.22
|
39.72
|
5.61
|
AsDMP32
|
37.00
|
15.86
|
40.97
|
6.17
|
AsDMP33
|
38.50
|
16.43
|
41.31
|
3.76
|
Phylogenetic analysis of DMP gene family in oat
To analyze the phylogenetic relationships of DMP family members among different species, we used the MEGA 11 eighbor-joining (NJ) method to construct a phylogenetic evolutionary tree based on 33 DMP members in oat and Arabidopsis, maize, rice and sorghum. The phylogenetic tree showed that the plant DMP gene family can be classified into five subfamilies, subfamilies I, II, III, IV and V (Fig. 3). Subclade V has the highest number of DMPs, containing 40 genes, of which 15 are AsDMPs. This was followed by subfamilies III, I, IV, and II, which contained 23, 15, 10, and 5 genes, respectively, of which 8, 7, 3, and 0 were AsDMPs, respectively. The oat DMP gene underwent significant expansion compared to other plants and the branching clustering pattern of the oat DMP protein was similar to that previously reported for cotton [17].
Conserved motifs of the oat DMP gene and analysis of gene structure
To further clarify the diversity and conservation of the oat DMP gene family during evolution, the conserved motifs, conserved domains and gene structures of AsDMP were analysed based on phylogeny (Fig. 4). A total of 10 Motifs were predicted through the MEME website (Fig. 4B; Fig. 5), with the number of motifs ranging from 4 to 9 for all DMP proteins, and all DMP members had Motif 3, Motif 4, and Motif 5, suggesting that these three motifs are highly conserved among the 33 AsDMP members. The basal composition and distribution of DMP members in the same subfamily are essentially the same, which may prove their functional similarity. It is noteworthy that there are differences in the Motif composition of some proteins in different or the same subfamily. For example, Motif 8 and Motif 10 are present for only some of the proteins in subfamily V, indicating their functional variability. In addition, certain motifs are absent in certain subfamily members, and some specific motifs are present only in specific genes. For example, Motif 1 and Motif 7 are missing in all AsDMP members in subfamily III, and Motif 7 is missing in subfamily IV. Motif 6 is present only in subfamily III, and Motif 8, Motif 9, and Motif 10 are also present in only some members. Whether the presence or absence of these specific motifs confers unique functional roles on the DMP genes requires further study, and the variation in subfamily motif composition may be due to their functional diversity. According to gene structure, no intron structure was found in other oat DMP members except AsDMP4 of subfamily I and AsDMP24 of subfamily V (Fig. 4D), indicating that although DMP family members may perform similar functions, there are still differences among individuals.
Chromosome localization, collinear analysis and Ka/Ks selection pressure analysis
In order to better understand the distribution mechanism of DMPs gene in oat chromosomes, the chromosome map of 33 DMP genes in oat was constructed according to the genome sequence of oat (Fig. 6), and the 33 identified AsDMP genes were distributed on 17 chromosomes. Five genes were distributed on each of chromosomes chr 3C and chr 4D, three genes on chr 4A and chr 6A, two genes on chr 1A, chr 2D, chr 3D, and chr 4C, and one gene on each of the remaining chromosomes. Tandem replication occurs on chr 3C, chr 4D, and chr 6A.
Gene family evolution mainly includes whole genome replication, fragment replication and tandem replication [25]. Most plants underwent an ancient genome-wide replication event, or polyploidy, resulting in the duplication of all genes in a region [26]. This large-scale chromosomal doubling event resulted in the retention of a large number of chromosomal doubling fragments in the genome [27]. Tandem repeats occur on the same chromosome and are adjacent to each other, often with similar sequences and similar functional clusters [28]. Fragment duplications are duplicated genes that located far apart or on different chromosomes. Gene duplication events are the main cause of gene family expansion and doubling [29, 30].
Through homology analysis of oat DMP genes, we visualized the relationships among oat DMP genes to elucidate the expansion mechanism of oat DMP gene family (Fig. 7). Among the 35 homologous gene pairs, 4 gene pairs were identified to have tandem replication events (AsDMP11/AsDMP12, AsDMP20/AsDMP21, AsDMP23/AsDMP24, AsDMP28/AsDMP29) (Fig. 6). Thirty-one pairs of genes underwent genome-wide replication or fragment replication. Therefore, we hypothesized that the major causes of gene amplification during DMP gene evolution were whole-genome duplication events or fragment duplication events.
In order to further understand the evolutionary relationship of AsDMP genes, we constructed collinearity maps of DMP families in oats, sorghum and maize (Fig. 8). 14 AsDMP genes were found to be colinear with at least two or more homologous genes, 19 pairs of colinear relationships between 14 AsDMPs and 5 SbDMPs, 14 pairs of colinear relationships between 14 AsDMPs and 4 ZmDMPs, and the 14 AsDMP genes with colinear relationships in sorghum and maize were the same, suggesting that poaceae family showed higher conservation between them. It suggests that these genes may have played an important role in evolution by participating more frequently in gene duplication events.
Selection pressure analysis was performed by judging the ratio of the number of nonsynonymous substitutions per nonsynonymous site (Ka) to the number of synonymous substitutions per synonymous site (Ks) (Ka/Ks)(Table 3). Not a Number (NaN) means that these pairs of genes have almost synonymous mutations at sites where synonymous mutations can occur, indicating that the sequence divergence is quite large and the evolutionary distance is very far. We found that since the Ks value of AsDMP-1/AsDMP-9, AsDMP-1/AsDMP-14, AsDMP-2/AsDMP-9, AsDMP-2/AsDMP-14, AsDMP-3/AsDMP-9, AsDMP-3/AsDMP-14 is NaN, The Ka/Ks is NaN. Among other gene pairs with segmental duplication, 22 gene pairs have Ka/Ks ratios less than 0.5, 3 gene pairs have Ka/Ks ratios between 0.5 and 1.0, and Ka/Ks are all less than 1, and 4 tandemly repeated genes The Ka/Ks ratio is also less than 1, indicating that these AsDMP genes were subject to purifying selection during the evolution process and functional differentiation occurred after whole-genome duplication/segment duplication.
Table 3
Ka/Ks values of duplicated DMP gene pairs from oat species
Gene name | Gene name | Duplication type | Ka | Ks | Ka/Ks |
AsDMP1 | AsDMP2 | WGD/semental | 0.027307 | 0.168114 | 0.16243 |
AsDMP1 | AsDMP3 | WGD/semental | 0.01553 | 0.027602 | 0.562622 |
AsDMP1 | AsDMP9 | WGD/semental | 0.219878 | NaN | NaN |
AsDMP1 | AsDMP14 | WGD/semental | 0.343854 | NaN | NaN |
AsDMP2 | AsDMP3 | WGD/semental | 0.017651 | 0.137493 | 0.128376 |
AsDMP2 | AsDMP9 | WGD/semental | 0.220654 | NaN | NaN |
AsDMP2 | AsDMP14 | WGD/semental | 0.345933 | NaN | NaN |
AsDMP3 | AsDMP9 | WGD/semental | 0.219878 | NaN | NaN |
AsDMP3 | AsDMP14 | WGD/semental | 0.34523 | NaN | NaN |
AsDMP4 | AsDMP18 | WGD/semental | 0.037855 | 0.0854 | 0.443265 |
AsDMP4 | AsDMP28 | WGD/semental | 0.046803 | 0.114023 | 0.410471 |
AsDMP5 | AsDMP31 | WGD/semental | 0.037644 | 0.103329 | 0.364314 |
AsDMP5 | AsDMP33 | WGD/semental | 0.029288 | 0.152551 | 0.191988 |
AsDMP6 | AsDMP27 | WGD/semental | 0.043386 | 0.064124 | 0.676584 |
AsDMP6 | AsDMP30 | WGD/semental | 0.038638 | 0.081658 | 0.473172 |
AsDMP7 | AsDMP8 | WGD/semental | 0.035414 | 0.089984 | 0.393563 |
AsDMP7 | AsDMP13 | WGD/semental | 0.003156 | 0.063771 | 0.049493 |
AsDMP8 | AsDMP13 | WGD/semental | 0.033273 | 0.109512 | 0.303831 |
AsDMP10 | AsDMP22 | WGD/semental | 0.059639 | 0.350143 | 0.170326 |
AsDMP11 | AsDMP17 | WGD/semental | 0.032496 | 0.128036 | 0.253804 |
AsDMP11 | AsDMP23 | WGD/semental | 0.037483 | 0.125291 | 0.29917 |
AsDMP15 | AsDMP20 | WGD/semental | 0.031438 | 0.084768 | 0.370873 |
AsDMP15 | AsDMP32 | WGD/semental | 0.0463 | 0.25935 | 0.178524 |
AsDMP17 | AsDMP23 | WGD/semental | 0.016389 | 0.050315 | 0.325726 |
AsDMP18 | AsDMP28 | WGD/semental | 0.012423 | 0.04846 | 0.25635 |
AsDMP19 | AsDMP25 | WGD/semental | 0.011719 | 0.100388 | 0.116737 |
AsDMP19 | AsDMP26 | WGD/semental | 0.011714 | 0.077808 | 0.150555 |
AsDMP20 | AsDMP32 | WGD/semental | 0.052084 | 0.305306 | 0.170598 |
AsDMP25 | AsDMP26 | WGD/semental | 0 | 0.034058 | 0 |
AsDMP27 | AsDMP30 | WGD/semental | 0.069509 | 0.108539 | 0.64041 |
AsDMP31 | AsDMP33 | WGD/semental | 0.012844 | 0.069892 | 0.183765 |
AsDMP11 | AsDMP12 | Tendem | 0.106861 | 0.351275 | 0.304209 |
AsDMP20 | AsDMP21 | Tendem | 0.060096 | 0.281151 | 0.21375 |
AsDMP23 | AsDMP24 | Tendem | 0.11315 | 0.303374 | 0.372974 |
AsDMP28 | AsDMP29 | Tendem | 0.026374 | 0.119097 | 0.221446 |
Cis-element analysis of AsDMP gene promotors
Gene expression was usually regulated by cis-elements in its upstream promoter sequence. Exploring the cis-regulatory elements contained in the promoter region of the oat DMP gene will help to understand the regulatory mechanism of the DMP gene and speculate on its potential functions. We use the PlantCARE database to conduct predictive analysis of promoter cis-regulatory elements. Based on the role and function of cis-regulatory elements, they are divided into the following categories: Hormone responsive elements include auxin responsive elements, gibberellin responsive elements, abscisic acid (ABA) responsive elements, salicylic acid responsive elements and MeJA responsive elements. Abiotic stress response elements include low temperature, defense and stress response elements, MYB binding sites (MYBHv1, light response, drought and flavonoid biosynthetic gene regulation), anaerobic induction and hypoxia-specific induction response elements. Growth and development-related regulatory elements include light-responsive elements, meristem expression-related elements, phytochrome-responsive elements, circadian rhythm control elements, cell cycle regulatory elements, and endosperm expression and seed-specific regulatory elements. Other responsive elements include AT-DNA (ATBP-1) binding sites, protein binding sites, activator mediated activation elements, and regulatory elements of zein metabolism.
Among the cis-elements involved in plant growth and development, light-responsive elements are the most abundant, with all except AsDMP10 having light-responsive elements and widely distributed in the promoter region. Elements related to endosperm expression are located in the AsDMP13, AsDMP15, AsDMP16, AsDMP21, AsDMP22, AsDMP29, AsDMP32, and AsDMP33 promoter regions; The seed-specific regulatory elements involved are located in the promoter regions of AsDMP5, AsDMP6, AsDMP7, AsDMP8, AsDMP11, AsDMP12, AsDMP13, AsDMP15, AsDMP17, AsDMP20, AsDMP21, AsDMP23, AsDMP24, AsDMP27, AsDMP29, AsDMP31 and AsDMP33. The regulatory elements involved in endosperm expression and seed-specific regulatory elements may be involved in the regulation of plant endosperm and seed development, suggesting that these DMP genes may play an important role in plant reproductive development.
Almost all promoters contain several hormone response elements, but the hormone response elements are not closely related to their subfamilies (Fig. 9). Among them, cis-acting elements involved in MeJA response were most abundant in the AsDMP gene promoter region, and 32 AsDMP gene promoter regions contained at least one MeJA response element. ABA-responsive elements are also widely present in the promoter region of AsDMP gene, with 29 AsDMP family members containing at least one ABA responsive element in their promoters. Stress related cis-regulatory elements are also widely distributed in the promoter region of AsDMP gene. Among the cis-acting elements of abiotic stress, MYB elements are the majority. Most AsDMP members contain one or more MYB elements in their promoters, which are involved in drought, photoresponse and the regulatory response of flavonoid biosynthesis genes. There are 20 AsDMP genes that respond to low temperature, and 13 AsDMP genes that respond to defense and stress. In addition, there are some cis-acting elements that are necessary for hypoxia and anaerobic induction. These results indicate that the transcription of oat AsDMP gene may be affected by multiple environmental factors.
Expression patterns of DMP gene in Oat at different tissue and developmental stages
Use of oat transcriptome data to analyse the expression pattern of the AsDMP gene in different tissues or at different developmental periods in the same tissue, including spikes, roots and leaves during seedling development, and seeds at the early, middle and late stages of development (Fig. 10). According to the transcriptome results, we found that AsDMP5, AsDMP6, AsDMP31, and AsDMP33 were not expressed in the above tissues. In AsDMP1, AsDMP2, AsDMP3 and AsDMP29, AsDMP3 was not expressed in leaves, and AsDMP29 was not expressed in ears and leaves, but was expressed in other tissues. And the expression of AsDMP1 and AsDMP3 was first down-regulated and then up-regulated in the early, middle and late stages of seed development, while AsDMP2 was consistently up-regulated and AsDMP29 was consistently down-regulated; AsDMP4 is expressed in seeds only at mid-development and not at early or late developmental stages; AsDMP7 and AsDMP8 were only expressed in spikes, suggesting that they may be related to spike development or fruit formation; AsDMP27 was expressed only in roots, and it was hypothesised that it might be involved in root growth and development. AsDMP9, AsDMP10, AsDMP13, AsDMP14, AsDMP15, AsDMP16, AsDMP22, AsDMP28, and AsDMP30 were each expressed during seed development; AsDMP19, AsDMP25, and AsDMP26 were expressed in spikes, roots, and leaves as well as in the early, middle, and late stages of seed development, and were first up-regulated and then down-regulated with the period of seed development. Notably, the expression of AsDMP25 and AsDMP26 was the highest among the 33 AsDMP genes in the middle stage of seed development, when the expression of these two genes was 4.2-fold and 2.3-fold higher than that in the early stage and 9-fold and 4.7-fold higher than that in the late stage. In summary, it is hypothesised that of the 17 genes expressed during seed development, 7 genes (AsDMP1, AsDMP2, AsDMP3, AsDMP19, AsDMP25, AsDMP26, AsDMP29) are involved in the whole process of seed development; 2 genes (AsDMP4, AsDMP14) are involved in mid-seed development; and 4 genes (AsDMP9, AsDMP13, AsDMP15, AsDMP30) are involved in late seed development.
Expression analysis of DMP gene in response to high temperature and aging of oat seeds
In order to further analyze the expression of oat DMP genes under natural aging and artificial high temperature aging, based on transcriptome analysis and according to the expression changes of AsDMP genes during seed development period, we selected 10 genes in the AsDMP gene family (AsDMP1, AsDMP2, AsDMP3, AsDMP10, AsDMP19, AsDMP22, AsDMP25, AsDMP26, AsDMP28, AsDMP29) were analyzed by qPCR (Fig. 11). The qPCR results showed that the expression of AsDMP1 and AsDMP19 was up-regulated in both natural aging treatment and artificial aging treatment, while the expression of AsDMP22 was down-regulated in both aging ways. It shows that the two genes AsDMP1 and AsDMP19 are involved in regulating the senescence and vigor reduction of oat seeds under natural aging and artificial aging, while the AsDMP22 gene plays a positive role in delaying or hindering the senescence of oat seeds under natural aging conditions and artificial high-temperature aging. It is worth noting that the AsDMP28 and AsDMP29 genes are only expressed under artificial aging treatment, indicating that the expression of AsDMP28 and AsDMP29 is induced under high temperature treatment, but not under natural aging.