Cloning and bioinformatics analysis of DXS, DXR, 10HGO, G10H and GPPS
We cloned DXS, DXR, 10HGO, G10H and GPPS genes from R. glutinosa and registered them in GenBank, DXS (MG764508), DXR (MG764509), 10HGO (MH102394), G10H (MK559439), GPPS (MG770219), their cDNA length is 2380bp, 1751 bp, 1192 bp, 2020 bp, 1314 bp,CDS length is 2154 bp, 1479 bp, 1065 bp, 1473 bp, 1272 bp, respectively. DXS has 8 exons and 7 introns, and none of the other genes have introns(Table 1,Fig. S1). The analysis of protein physical and chemical properties showed that the molecular weight (MW) of DXS, DXR, 10HGO, G10H and GPPS proteins were 77.12KD, 53.48 KD, 38.44 KD, 55.21 KD, 46.34 KD,PI were 6.80,5.79,6.21,8.81,6.04 respectively, and DXR and 10HGO were hydrophobic. The secondary structure prediction shows that the α helix ratio is 24.01–57.82%, and the β-folding ratio is 3.55–8.94%. The prediction of subcellular localization showed that 10HGO was located in the cytoplasm and the others were located in the chloroplast(Table 2).
Table 1
Sequence information of DXS、DXR、10HGO、G10H、GPPS synthase genes in R. glutinosa
Gene Name
|
Accession Number
|
cDNA Length (bp)
|
CDS Length (bp)
|
No. of Extron
|
DXS
|
MG764508
|
2380
|
2154
|
8
|
DXR
|
MG764509
|
1751
|
1479
|
1
|
10HGO
|
MH102394
|
1192
|
1065
|
1
|
G10H
|
MK559439
|
2020
|
1473
|
1
|
GPPS
|
MG770219
|
1314
|
1272
|
1
|
Table 2
The analysis of DXS/DXR/10HGO/G10H/GPPS proteins in R. glutinosa
Protein Name
|
No.of AA
|
Physico-chemical Property
|
Secondary Structure
|
Subcellular location
|
MW (kD)
|
PI
|
hydrophilicity
|
Alpha helix (%)
|
Extended strand (%)
|
Beta turn (%)
|
Random coil (%)
|
DXS
|
717
|
77.12
|
6.80
|
hydrophilcity
|
38.77
|
15.62
|
6.42
|
39.19
|
Chloroplast
|
DXR
|
492
|
53.48
|
5.79
|
hydrophobicity
|
34.35
|
19.92
|
8.94
|
36.79
|
Chloroplast
|
10HGO
|
354
|
38.44
|
6.21
|
hydrophobicity
|
24.01
|
25.42
|
7.34
|
43.22
|
Cytosol
|
G10H
|
491
|
55.21
|
8.81
|
hydrophilcity
|
51.32
|
12.42
|
6.92
|
29.33
|
Chloroplast
|
GPPS
|
422
|
46.34
|
6.04
|
hydrophilcity
|
57.82
|
4.98
|
3.55
|
33.65
|
Chloroplast
|
AA represents amino acid, PI represents theoretical isoelectric point, MW represents Molecular weight. |
Using NCBI database to compare plant homologous proteins. The amino acids encoded by RgDXS have more than 90% similarity with Salvia splendens, Sesamum indicum, Salvia officinalis. The amino acids encoded by RgDXR have more than 80% similarity with Handroanthus impetiginosus, Osmanthus fragrans, Olea europaea var. sylvestris. The amino acid encoded by Rg10HGO, RgG10H and RgGPPS has the highest similarity with Striga asiatica(80.74%), Phtheirospermum japonicum(92.81%) and Sesamum indicum(90.07%),respectively(Fig. S2). In addition,build the Phylogenetic tree of DXS, 10HGO, G10H and GPPS, R.glutinosa and Scutellaria barbata, Striga asiatica, Handroanthus impetiginosus, Sesamum indicum,were clustered into one branch, respectively, and DXR branches separately(Fig. S3).
Based on STRING genome, gene co-ocurrence on DXS,DXR,10HGO,G10H,GPPS synthase of R. glutinosa was analyzed, found that they were conservative in organisms, occurred in Archaea, Eukaryota and Bacteria, in which they were more conservative in Viridiplantae. Many genes in Viridiplantae appeared to match DXS,DXR,10HGO,G10H,GPPS synthase of R. glutinosa, gene co-ocurrence for DXS,DXR,10HGO,G10H,GPPS of R. glutinosa, were all found in 19 species of plants, such as Aquilegia coerulea,Citrus clementine,Citrus sinensis,etc. 4 synthase genes were respectively matched from 9 species of plants, 3 synthase genes were respectively matched from 34 species of plants, and only 1–2 synthase genes in 10 species of plants had co-ocurrence, and mainly belong to GPPS.The co-occurrence ratios of DXS, DXR, 10HGO, G10H and GPPS were 87.5%, 94.4%, 38.9%, 30.6% and 90.3%, respectively(Table S2). Further analysis showed that DXS, DXR, 10HGO, G10H and GPPS had high homology in eukaryotes (Table S3,Fig. S4a,b),it has the highest homology with Erythranthe guttata, and also has high homology with Aquilegia coerulea, Citrus clementina, Citrus sinensis and Gossypium raimondii.
Expression of DXS, DXR, 10HGO, G10H and GPPS
DXS
In stage I-IV, the expression of DXS in Jinju roots was significantly higher than that in 85 − 5 roots, then was basically the same at stage V, especially in stage IV, Jinjiu expression level was 2-fold that of 85 − 5. In the stage I-IV, the expression of DXS in Jinjiu leaves was significantly higher than that in 85 − 5 leaves, especially in stage IV, Jinjiu expression level was 13-fold that of 85 − 5, but in V stage was lower than 85 − 5 leaves. The overall expression in leaves was higher than that in roots(Fig. 1a).
DXR
In stage I-III and V, the expression of DXR in Jinjiu root was higher than that in 85 − 5 root, but in stage IV was significantly lower than that in 85 − 5 root. In stage IV-V(mature stage), DXR in Jinjiu leaf was significantly higher than that in 85 − 5 leaf. The expression level in the leaves of stage IV-V(mature stage) was higher than that in the roots(Fig. 1b).
10HGO
In stage II-V, the expression of 10HGO in Jinjiu root was lower than that in 85 − 5, especially in stage V, 85 − 5 expression level was 2-fold that of Jinjiu. In stage IV-V (mature stage), the expression of 10HGO in Jinjiu leaf was significantly higher than that in 85 − 5 leaf, especially in stage V, Jinjiu expression level was 2-fold that of 85 − 5(Fig. 1c).
G10H
In stage I-IV, the expression of G10H in Jinjiu root was higher than that in 85 − 5 roots, especially in stage III, Jinjiu expression level was 10-fold that of 85 − 5. The expression change in leaves was more complex, In stage IV, the expression of G10H in Jinjiu leaves was significantly higher than that in 85 − 5 leaves, while in V stage, lower than that in 85 − 5 leaves. The expression level in leaves was significantly higher than that in roots(Fig. 1d).
GPPS
In stage II-V, the expression of GPPS in Jinjiu root was lower than 85 − 5, especially in stage I V, 85 − 5 expression level was 3-fold that of Jinjiu. The expression of GPPS in Jinjiu leaf was significantly lower than that in 85 − 5 leaf except for stage IV(Fig. 1e).
On the whole, gene expression showed variety specificity, tissue specificity and spatio-temporal specificity, in stage IV-V, genes expression is at a higher level in the whole growth cycle, usually reaching a peak in stage IV or V. The expression of DXS, DXR and G10H genes in Jinjiu roots and leaves was more than 85 − 5, while the expression of 10HGO and GPPS genes in Jinjiu roots and leaves was less than 85 − 5, with the maturity of R.glutinosa, DXS and G10H expression showed a trend of increasing at first and then decreasing as a whole, and decreased significantly in stage V, DXR 10HGO and GPPS expression decreased at first and then increased, and often continued to increase in stage IV-V.
Clustering heat map analysis
In order to study the expression level of genes in 85 − 5 roots, 85 − 5 leaves, Jinjiu roots and Jinjiu leaves in the same period, the expression of DXS in 85 − 5 roots was used as control, the expression of each gene was calculated and cluster heat map was drawn,so that to study genes clustering expression.
In stage I, the expression of DXS and G10H in leaves was higher than roots, Jinjiu leaves was higher than 85 − 5 leaves, Jinjiu roots was higher than 85 − 5 roots, while the expression of 10HGO, DXR and GPPS in roots was higher than that in leaves (Fig. 2a), cluster analysis showed that the expressions of 10HGO, DXR and GPPS genes were grouped into one group, DXS and G10H were clustered into one group. In stage II, the expression of DXS and G10H in leaves was higher than that in roots, and the expression of DXR, 10HGO and GPPS in 85 − 5 was higher than that in Jinjiu, cluster analysis showed that the expression of 10HGO, DXR and GPPS genes were clustered into one group, and the expression of DXS and G10H were clustered together (Fig. 2b), and the clustering was the same as that of stage I. In stage III, the expression of DXS and G10H in leaves was higher than that in roots, Jinjiu leaves was higher than 85 − 5 leaves, Jinjiu roots was higher than 85 − 5 roots, DXR in Jinjiu was higher than that in 85 − 5, and 10HGO and GPPS in 85 − 5 was higher than that in Jinjiu (Fig. 2c),cluster analysis showed that the expression of DXS, DXR and G10H was grouped into one group, and 10HGO and GPPS were grouped into one group. In stage IV, the expression of DXS, DXR, 10HGO, G10H and GPPS in leaves was higher than that in roots, and Jinjiu leaves was higher than 85 − 5 leaves, 85 − 5 roots was higher than Jinjiu roots(Fig. 2d),cluster analysis showed that the expression of 10HGO, DXR and G10H was clustered into one group, DXS and GPPS was clustered into one group. In stage V, the expression of DXS, DXR, 10HGO, G10H and GPPS in leaves was higher than that in roots, and the expression was more complex, cluster analysis showed that DXR and GPPS gene expression were clustered into one group, DXS and G10H gene expression were clustered into one group, and 10HGO was a separate group(Fig. 2e).
Accumulation dynamics of iridoid glycosides
Different varieties
In roots, the content of total iridoid glycosides of 85 − 5 and Jinjiu reached the highest in stage IV and decreased in stage V, and in the mature stage of R.glutinosa (IV-V), the content of iridoterpene glycosides in Jinjiu roots was significantly higher than that in 85 − 5 roots. In leaves, the content of total iridoid glycosides of 85 − 5 and Jinjiu reached the highest in stage III and decreased continuously in stage IV-V, in mature stage (IV-V stage), Jinjiu leaves was significantly lower than that in 85 − 5 leaves. Further comparison showed that the content of total iridoid glycosides in leaves was higher than that in roots in the early growth stage (I-II stage), from the middle growth stage (III stage), the content of roots was higher than that in leaves, and it was also the same in the later growth stage (IV-V stage) (Fig. 3a). It is worth noting that the content of iridoid glycosides in Jinjiu root was significantly higher than that in 85 − 5 roots in the mature stage(IV-V stage).
Different producing areas
In roots, the content of total iridoid glycosides in Wenxian was the highest in stage III, and that in Xinxiang was the highest in stage IV, in the mature stage (IV-V stage), the content of total iridoid glycosides in Wenxian root was lower than that in Xinxiang root. In leaves, the content of total iridoid glycosides in Wenxian and Xinxiang reached the highest in stage III, decreased continuously in stage IV-V, and in mature stage (IV-V), Wenxian leaves was slightly lower than that in Xinxiang leaves. Further comparison showed that in the early growth stage (I-II stage), the content of total iridoid glycosides in leaves was higher than that in roots, but from the middle growth stage (III stage), Wenxian root increased significantly, then in stage IV, Xinxiang root increased significantly, but both decreased in stage V (Fig. 3b).It is worth noting that in stage V, the content of iridoid glycosides in Wenxian root was almost the same as that in Xinxiang root.
Analysis of the correlation between iridoid glycoside accumulation and gene expression
R.glutinosa uses roots as medicine, we use Jinjiu root and 85 − 5 root as research materials, SPSS17.0 software to analyze the correlation between iridoid glycoside accumulation and related enzyme genes expression.The results showed that there was a significant negative correlation between the content of total iridoid glycosides and the expression of GPPS in 85 − 5 roots, and the correlation coefficient was − 0.906(P༜0.05), and there was no significant correlation with DXS, DXR, 10HGO and G10H. In Jinjiu roots, the content of total iridoid glycosides was significantly negatively correlated with the expression of DXR, and the correlation coefficient was − 0.991(P༜0.01), and there was no significant correlation with DXS, GPPS, 10HGO and G10H (Table S4).
Level of Genomic Methylation in R. glutinosa
With the growth of R.glutinosa, the content of 5mC in roots and leaves reached the maximum in stage III, and decreased continuously in stage IV-V, and the content of 5mC in leaves was higher than that in roots during the whole growth cycle (Fig. S5). Without changing the methylation status of the restriction site, the methylation state of DNA sequence can be detected by using HpaII and MspI with different sensitivity to CCGG methylation. HpaII is not sensitive to the full methylation (double-stranded methylation) of single or two cytosines and only cleaves hemimethylation, while MspI is sensitive to fully methylated internal cytosine and insensitive to fully methylated external cytosine. Therefore, the MSAP analysis of the restriction fragment can reflect the degree and status of methylation of the enzyme site. In this study, the band patterns measured by MSAP method are divided into I, II, III and IV, 4 species. I: Both H and M have bands and no methylation occurs (Fig. S6a); II: HpaII has bands and MspI no bands, external methylation of single-stranded DNA (Fig. S6b); III: HpaII no bands and MspI has bands, internal methylation of double-stranded DNA (Fig. S6c); IV: MSAP could not detect the complete methylation sites of cytosine (mCCGG/GGCmC or mCmCGG/GGmCmC) on the outside or inside and outside of DNA, so H and M had no bands at this site, and the band pattern in this case was expressed by IV.
We used two groups of restriction endonuclease digestion combinations of EcoRI and MspI, EcoRI and HpaII, among the 48 pairs of primer combinations, 6 pairs of primer combinations were selected, resulting in 39 polymorphic loci, and the bands were statistically analyzed after selective amplification.
In roots, six pairs of primers amplified about 60–84 clear bands, of which the number of methylated bands was 24–106, accounting for 44.44%-60.00%, and the proportion of total methylation bands was 20.99%-27.14%, indicating that most of the genomic CCGG sequences in R.glutinosa roots are in a state of hemimethylation and full methylation (Table S5). In leaves, six pairs of primers amplified about 73–99 clear bands, of which the number of methylated bands was 40–56 bands, accounting for 46.46%-60.22%, and the proportion of total methylation bands was 21.21%-30.11%, indicating that most of the genomic CCGG sequences in R.glutinosa leaves are in a state of hemimethylation and full methylation (Table S6). Further analysis showed that the total methylation ratio of roots reached the highest in stage II, reaching 27.14%, and stages II, IV and V were basically the same (27.14%, 26.87%, 26.67%), and the ratio of leaves reached the highest in stage V, reaching 30.11%, both roots and leaves show the trend of decreasing first and then increasing (Fig. 4a). The ratio of methylated bands of roots reached the highest in stage V, reaching 60.00%, and there was little difference in II-V (58.57%, 58.33%, 56.72%, 60.00%), and the ratio of leaves reached the highest in stage V, reaching 60.22%, and there was little difference between stage IV and V (57.53%, 60.22%), both roots and leaves show the trend of decreasing first and then increasing (Fig. 4b).
Effects of 5-azaC on accumulation of iridoid glycosides in R.glutinosa
The content of total iridoid glycosides in 85 − 5 roots accumulated continuously with the increase of growth period, after treatment with 5-azaC, content of iridoid glycosides increased significantly, in the same growing period, the content of total iridoid glycosides increased with the increase of 5-azaC concentration, especially in the mature stage (IV-V), high concentration is more significant (100µM or 250µM). It is worth noting that in stage III, 250µM 5-azaC significantly inhibited the synthesis of iridoid glycosides(Fig. 5a). The content of total iridoid glycosides in 85 − 5 leaves was more complicated. In stage I, II, III, V, different concentrations of 5-azaC had no significant effect on the accumulation of total iridoid glycosides, only in stage IV, 5-azaC has obvious promotion effect. It is worth noting that in stage II and V, low concentration of 5-azaC (15µM-100µM) significantly inhibited the synthesis of iridoid glycosides (Fig. 5b).
In Jinjiu roots, 5-azaC promoted the accumulation of iridoid glycosides during the whole growth cycle, in the early growth stage (I-II stage) of R.glutinosa, the accumulation of iridoid glycosides at different concentrations of 5-azaC was very significant, but in the middle growth stage (III stage) showed inhibitory effect, then in the late growth stage (IV-V stage), it showed a promoting effect but was not significant (Fig. 5c). The content of total iridoid glycosides in Jinjiu leaves was more complicated. In general, 5-azaC promoted the accumulation of iridoid glycosides,and in stage III, IV, V, promoting effect of low concentration and high concentration was significant, but middle concentration was not significant (Fig. 5d).
Effects of 5- 5-azac on gene expression in R.glutinosa
After treatment with different concentrations of 5-azaC, we detected the changes of DXS, DXR, 10HGO, G10H and GPPS expression.
In 85 − 5 roots, 5-azaC promoted the expression of DXS, DXR, 10HGO, G10H and GPPS in varying degrees, with the increase of 5-azaC concentration, the promoting effect increased at first and then decreased, the promoting effect of 15–50µM 5-azaC was better than other concentration, when the concentration of 5-azaC was more than 100µM, it inhibited the expression as a whole. It is worth noting that the expression of G10H in stage IV, 30µM 5-azaC treatment was nearly 5 times higher than that in the control group, and that of GPPS in stage III, 50µM 5-azaC treatment was nearly 6 times higher than that in the control group (Fig. 6a). In Jinjiu roots, different concentrations of 5-azaC significantly inhibited the expression of DXS, which decreased at first and then enhanced with the increase of concentration, and 5-azaC promoted the expression of DXR, 10HGO and GPPS to a certain extent, especially in the stage I-II, both low concentration (15µM, 30µM) and high concentration (100µM, 250µM) significantly promoted the expression. 5-azaC promoted the expression of G10H in varying degrees, with the increase of 5-azaC concentration, the promoting effect of G10H increased at first and then decreased, 30–50µM 5-azaC has great promoting effect, and when the concentration of 5-azaC was more than 100µM, inhibition of expression. It is worth noting that in stage IV, the expression of DXS under different concentrations of 5-azaC was more than 10 times lower than that of the control group, the expression of DXR in stage II, 250µM 5-azaC was nearly 6 times higher than that of the control group, and the expression of G10H in stage II, 50µM 5-azaC treatment was nearly 10 times higher than that of the control group(Fig. 6b).
In 85 − 5 leaves, 5-azaC promoted the expression of DXS, DXR, 10HGO, G10H and GPPS in varying degrees, and the promoting effect increased at first and then decreased with the increase of 5-azaC concentration, and promoting effect of 15–50µM 5-azaC was better, when the concentration of 5-azaC was more than 100µM, the promoting effect was not as significant as that of low concentration. It is worth noting that the expression of GPPS in stage III, 50µM 5-azaC treatment was nearly 6 times higher than that of the control group, while that of G10H in stage II, treated with various concentrations of 5-azaC was more than 4 times lower than that of the control group, and in stage IV, the expression level of G10H treated with 30µM 5-azaC was 5 times lower than that of the control group (Fig. 6c). In Jinjiu leaves, 5-azaC promoted the expression of DXS, DXR, 10HGO, G10H and GPPS in varying degrees, with the increase of 5-azaC concentration, the promoting effect increased at first and then decreased. The promoting effect of 15–50µM 5-azaC was better, when the concentration of 5-azaC was more than 100µM, the promoting effect was not as significant as that of low concentration. It is worth noting that the expression of DXS in stage III and IV was lower than that in the control group under all concentrations of 5-azaC, while the promoting effect of DXR in stage II was very significant, especially at 30µMand 100µM, it increased by 14 and 25 times, respectively (Fig. 6d).
Effects of 5-azaC on 5mC Content in Genomic DNA of R. glutinosa
We used different concentrations of 5-azaC to treat roots and leaves, and found that with the maturity of R.glutinosa, the content of genomic 5mC in roots and leaves in mature stage was significantly higher than that in seedling stage, and the content of genomic DNA 5mC in roots and leaves decreased with the increase of 5-azaC concentration, 5-azaC significantly inhibited the formation of genomic methylation in roots and leaves (Fig. 7a,b). In different growth periods, the content of 5mC in the control group was the highest, and the inhibitory effect gradually enhanced with the increase of 5-azaC concentration in the same growth period, further analysis showed that the content of genomic 5mC in R.glutinosa root and leaf was different at the same period, and leaf was higher than that in root tuber.
Effect of 5-azaC on the level of Genomic Methylation
In roots, 6 pairs of primers amplified about 61–146 clear bands, of which the number of methylated bands was 31–80 bands, accounting for 45.63%-76.29%, and the proportion of fully methylated bands was 16.98%-39.02%, indicating that most of the genomic CCGG sequences in R.glutinosa roots are in a state of hemimethylation and total methylation (Table S7). The total methylation ratio of the control group was significantly lower than that of the treatment group except stage II, and increased at first and then decreased with the increase of 5-azaC concentration (Fig. 8a). The change of methylation band ratio (MSAP%) is more complex, MSAP% of the control group in stage I is slightly lower than that in the other four stages, and there is no significant difference in the other four stages, on the whole, MSAP% of 5-azaC treatment in the same growth period is basically higher than that of the control group, and the maximum value of 15µM-100µM concentration treatment appeared in stage IV, IV, IV and V, respectively, and the maximum value of 250µM concentration treatment appeared in stage II (Fig. 8b).
In leaves, 6 pairs of primers amplified about 61–130 clear bands, of which the number of methylated bands was 24–83 bands, accounting for 33.33%-68.03%, and the proportion of fully methylated bands was 13.04%-35.29%, indicating that most of the genomic CCGG sequences in R.glutinosa leaves are in a state of hemimethylation and total methylation (Table S8). The change of total methylation ratio in leaves was more complex, low concentration of 5-azaC in the same growth period played a promoting role, while high concentration of 5-azaC played an inhibitory role, the control group reached the maximum value in stage III, the maximum value of 15µM-100µM concentration treatment appeared in II, III, III and II stage, and the maximum value of 250µM concentration treatment appeared in V stage (Fig. 8c). Under the treatment of 5-azaC, in I-III stage, the methylation band ratio (MSAP%) was promoted at low concentration, inhibited at high concentration, but both inhibited in IV and V stage. The control group reached the maximum value in stage IV, and the maximum value of 15µM-250µM concentration treatment appeared in stage II, III, IV, II and III, respectively (Fig. 8d).
Change of Methylation Status in R. glutinosa treated with 5-azaC
We used roots and leaves of mature R.glutinosa to analyze changes in DNA methylation status. Compared with the control group, the changes of genomic methylation status of R.glutinosa treated with 5-azaC could be divided into 3 categories and 12 bands. We set class A as a monomorphic band, means that the control group has the same methylation sites as the treatment group, indicating that the methylation status of CCGG sites remains unchanged, while B and C are polymorphic bands, means that methylation sites in the control and treatment groups were different, type B is methylation increased band pattern, indicating that the methylation level of genomic DNA has increased, and the band pattern of DNA methylation increase can be summarized as follows: I→III, I→IV, II→III, II→IV. Type C is the demethylation type, which indicates that the methylation level of genomic DNA has decreased, and the band pattern of demethylation change is: IV→II, IV→I, III→II, III→I. Due to the limitation of MSAP technique, the increase or decrease of DNA methylation could not be determined as I→II, III→IV, IV→III, II→I.
Statistical analysis of genomic DNA methylation increases and decreases in the band pattern (Table 3), we found that in roots most of the bands with increased methylation were II→IV, which changed from semi-methylation to full methylation, and a few transformed bands were I→III, at the concentration of 30µM-100µM 5-azaC, the methylation band increased significantly. The demethylation band type is mainly IV→II, with the increase of 5-azaC concentration, the number of demethylated bands also increased, and reached the maximum value of 12 bands at 50µM, III→I and IV→I also account for a certain proportion of demethylated bands,and as the concentration increased, the number of demethylated bands also increased and all reached the maximum value at 250µM. Further analysis we found that the number of methylation sites decreased more than the number of methylation sites increased, which was consistent with the trend of MSAP% under 5-azaC treatment in stage V.
Table 3
DNA mathylation pattern of Rehmannia root under 5-azaC
Type of methylation
pattern
|
Band Type
|
Number of sites
|
CK
|
Treat
|
Root
|
|
H
|
M
|
H
|
M
|
CK-
15µM
|
CK-
30µM
|
CK-
50µM
|
CK-
100µM
|
CK-
250µM
|
B1(I→III)
|
1
|
1
|
0
|
1
|
0
|
2
|
1
|
1
|
0
|
B2(II→III)
|
1
|
0
|
0
|
1
|
1
|
0
|
0
|
0
|
0
|
B3(II→IV)
|
1
|
0
|
0
|
0
|
2
|
5
|
6
|
4
|
2
|
B4(I→IV)
|
1
|
1
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
C1(III→I)
|
0
|
1
|
1
|
1
|
3
|
3
|
6
|
8
|
9
|
C2(III→II)
|
0
|
1
|
1
|
0
|
0
|
0
|
0
|
1
|
0
|
C3(IV→II)
|
0
|
0
|
1
|
0
|
4
|
9
|
12
|
9
|
9
|
C4(IV→I)
|
0
|
0
|
1
|
1
|
0
|
0
|
2
|
7
|
8
|
The methylation increase band pattern and demethylation band pattern in leaves are more than those in roots (Table 4), we found that in leaves most of the bands with increased methylation were II→IV, which changed from semi-methylation to full methylation, and a few transformed bands were I→III, at the concentration of 30µM-50µM 5-azaC, the methylation band increased significantly. The demethylation band type is mainly IV→II, with the increase of 5-azaC concentration, the number of demethylated bands also increased, and reached the maximum value of 16 bands at 250µM, III→I, III→II and IV→I also account for a certain proportion of demethylated bands, and as the concentration increased, the number of demethylated bands also increased and all reached the maximum value at 250µM also. Further analysis we found that the number of methylation sites decreased more than the number of methylation sites increased, especially under high concentration treatment, the number of bands decreased significantly, which was consistent with the trend of MSAP% under 5-azaC treatment in stage V.
Table 4
DNA mathylation pattern of Rehmannia leaf under 5-azaC
Type of methylation
pattern
|
Band Type
|
Number of sites
|
CK
|
Treat
|
Leaf
|
|
H
|
M
|
H
|
M
|
CK-
15µM
|
CK-
30µM
|
CK-
50µM
|
CK-
100µM
|
CK-
250µM
|
B1(I→III)
|
1
|
1
|
0
|
1
|
0
|
3
|
2
|
2
|
0
|
B2(II→III)
|
1
|
0
|
0
|
1
|
1
|
0
|
0
|
0
|
0
|
B3(II→IV)
|
1
|
0
|
0
|
0
|
3
|
5
|
7
|
3
|
2
|
B4(I→IV)
|
1
|
1
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
C1(III→I)
|
0
|
1
|
1
|
1
|
4
|
4
|
8
|
9
|
10
|
C2(III→II)
|
0
|
1
|
1
|
0
|
0
|
0
|
0
|
1
|
0
|
C3(IV→II)
|
0
|
0
|
1
|
0
|
5
|
7
|
10
|
13
|
16
|
C4(IV→I)
|
0
|
0
|
1
|
1
|
0
|
0
|
3
|
6
|
8
|
In general, the number of methylated and demethylated bands increased with the increase of 5-azaC concentration in roots and leaves of R.glutinosa, and effects of 5-azaC on genomic methylation status of roots and leaves were roughly the same.