Identification of GLYI and GLYII genes in the B. napus genome
Glyoxalase proteins (GLYI and GLYII) had been identified in Brassicaceae plant A. thaliana and B. rapa. Thus, AtGLYI/AtGLYII and BrGLYI/BrGLYII sequences were used as quires to blast genome database of B. napus and the sequences with E-value under 1E-10 were screened out. Then proteins that included the glyoxalase domain (PF00903) and the metallo-beta-lactamase domain (PF00753) were classified as BnaGLYI and BnaGLYII proteins, respectively. In this study, thirty-five BnaGLYI and 30 BnaGLYII genes were identified in B. napus. Table 1 summarized the gene ID, chromosomal distribution, number of introns, CDS and amino acid length, predicted sub-cellular localization, calculated molecular weight, and isoelectric points of the putative BnaGLYI and BnaGLYII.
Table 1
Detail information of BnaGLYI genes identified in Brassica napus L. genomes
Gene Symbol
|
Locus Identifier
|
Location
|
Gene Start(bp)
|
Gene Stop(bp)
|
Strand
|
No. of Introns
|
CDS Length(bp)
|
PP Length(aa)
|
MW(kDa)
|
pI
|
Localization
|
BnaGLYI01
|
BnaA02g19970D
|
A02
|
12337272
|
12338438
|
-
|
2
|
504
|
168
|
18818.68
|
5.81
|
Cya; Nub
|
BnaGLYI02
|
BnaA03g10440D
|
A03
|
4700661
|
4701804
|
+
|
3
|
582
|
194
|
21785.91
|
8.43
|
Cya; Mtb
|
BnaGLYI03
|
BnaA05g35240D
|
A05
|
670044
|
672049
|
-
|
3
|
414
|
138
|
15252.22
|
5.46
|
Cya; Nub
|
BnaGLYI04
|
BnaA06g04170D
|
A06
|
2547123
|
2548306
|
-
|
3
|
516
|
172
|
19274.7
|
7.77
|
Cya; Chb
|
BnaGLYI05
|
BnaA06g04580D
|
A06
|
2708014
|
2710119
|
+
|
7
|
714
|
238
|
26635.17
|
8.33
|
Cya ;Oma;Chb,c
|
BnaGLYI06
|
BnaA06g07360D
|
A06
|
3915659
|
3923909
|
+
|
26
|
3648
|
1216
|
134984.62
|
8.86
|
Cya,b
|
BnaGLYI07
|
BnaA06g10060D
|
A06
|
5342612
|
5343786
|
+
|
2
|
525
|
175
|
19778.79
|
5.68
|
Cya; Chb
|
BnaGLYI08
|
BnaA07g13890D
|
A07
|
12263440
|
12263994
|
+
|
0
|
555
|
185
|
20887.37
|
4.78
|
Cya,b
|
BnaGLYI09
|
BnaA07g26290D
|
A07
|
19358162
|
19361477
|
-
|
8
|
1026
|
342
|
37894.44
|
6.48
|
Cya; Chb,c
|
BnaGLYI10
|
BnaA08g23870D
|
A08
|
16843513
|
16844540
|
-
|
2
|
525
|
175
|
19846.96
|
5.88
|
Cya; Chb
|
BnaGLYI11
|
BnaA08g25110D
|
A08
|
17357805
|
17359871
|
-
|
7
|
852
|
284
|
31872.41
|
5.26
|
Cya,b
|
BnaGLYI12
|
BnaA09g49270D
|
A09
|
32814199
|
32815336
|
+
|
2
|
414
|
138
|
15394.22
|
5.84
|
Cya; Chb
|
BnaGLYI13
|
BnaA09g49870D
|
A09
|
33093200
|
33095241
|
+
|
1
|
1323
|
441
|
48072.12
|
5.72
|
Cya ;Oma;Cyskb
|
BnaGLYI14
|
BnaA09g56790D
|
A09
|
3889968
|
3891223
|
+
|
2
|
504
|
168
|
18943.89
|
5.86
|
Cya; Chb
|
BnaGLYI15
|
BnaA10g04310D
|
A10
|
2272334
|
2273834
|
-
|
1
|
1338
|
446
|
48973.02
|
5.45
|
Oma; Cyb
|
BnaGLYI16
|
BnaA10g11070D
|
A10
|
9346288
|
9347612
|
-
|
3
|
588
|
196
|
21959
|
6.71
|
Cya; Mtb
|
BnaGLYI17
|
BnaC02g23290D
|
C02
|
20281012
|
20282133
|
-
|
2
|
504
|
168
|
18762.53
|
5.66
|
Cya; Nub
|
BnaGLYI18
|
BnaC02g46640D
|
C02
|
2394713
|
2395834
|
+
|
2
|
504
|
168
|
18804.61
|
5.66
|
Cya; Nub
|
BnaGLYI19
|
BnaC03g13130D
|
C03
|
6313042
|
6314254
|
+
|
3
|
582
|
194
|
21712.86
|
7.77
|
Cya; Chb
|
BnaGLYI20
|
BnaC03g51010D
|
C03
|
35479070
|
35480133
|
-
|
4
|
453
|
151
|
16992.13
|
5.25
|
Cya; Nub
|
BnaGLYI21
|
BnaC05g04530D
|
C03
|
2231485
|
2233489
|
-
|
1
|
1338
|
446
|
48857.97
|
5.65
|
Cya, b ;Oma
|
BnaGLYI22
|
BnaC05g05340D
|
C05
|
2609787
|
2610529
|
-
|
2
|
417
|
139
|
15596.41
|
5.94
|
Cya, b
|
BnaGLYI23
|
BnaC05g05770D
|
C05
|
2848240
|
2850316
|
+
|
7
|
708
|
236
|
26438.95
|
8.74
|
Cya; Pea;Chb,c
|
BnaGLYI24
|
BnaC05g08770D
|
C05
|
4659651
|
4665014
|
+
|
19
|
2175
|
725
|
80672.51
|
7.49
|
Cya, b ;Oma
|
BnaGLYI25
|
BnaC05g11680D
|
C05
|
6800739
|
6802181
|
+
|
2
|
525
|
175
|
19733.75
|
5.68
|
Cya; Chb
|
BnaGLYI26
|
BnaC06g28360D
|
C06
|
29576211
|
29578606
|
-
|
8
|
1038
|
346
|
38368.97
|
6.19
|
Cya;Chb,c
|
BnaGLYI27
|
BnaC08g15100D
|
C08
|
19658835
|
19661229
|
+
|
7
|
852
|
284
|
31814.27
|
5.26
|
Cya, b
|
BnaGLYI28
|
BnaC08g16660D
|
C08
|
20576966
|
20578018
|
+
|
2
|
525
|
175
|
19821.86
|
5.89
|
Cya; Chb
|
BnaGLYI29
|
BnaC08g38920D
|
C08
|
34930459
|
34931660
|
-
|
2
|
522
|
174
|
19568.62
|
5.86
|
Cya; Mtb
|
BnaGLYI30
|
BnaC08g44820D
|
C08
|
37849659
|
37851160
|
-
|
1
|
1323
|
441
|
48162.21
|
5.46
|
Cya; Cyskb
|
BnaGLYI31
|
BnaC09g53670D
|
C09
|
3644531
|
3644929
|
+
|
0
|
399
|
133
|
15196.72
|
7.77
|
Cya, b
|
BnaGLYI32
|
BnaCnng38880D
|
Cnn
|
37521585
|
37522696
|
-
|
2
|
414
|
138
|
15502.36
|
6.2
|
Cya; Chb
|
BnaGLYI33
|
BnaCnng47290D
|
Cnn
|
46764251
|
46765432
|
-
|
5
|
432
|
144
|
16180.32
|
5.39
|
Cya, b
|
BnaGLYI34
|
BnaCnng47280D
|
Cnn
|
46758036
|
46759221
|
-
|
5
|
324
|
108
|
12264.1
|
5.01
|
Cya; Nub
|
BnaGLYI35
|
BnaCnng59150D
|
Cnn
|
58862580
|
58863862
|
-
|
3
|
414
|
138
|
15253.16
|
5.45
|
Cya; Nub
|
Abbreviations: CDS: coding DNA sequence, PP: polypeptide length, MW: molecular weight, PI: isoelectric point, bp: base pair, aa: amino acid, kDa: kilodalton, Ch: chloroplast, Cy: cytosol, Mt: mitochondria, Nu: nucleus, Pe: periplasm, Ou: outermembrane, Ec: extracellular matrix. |
a Localization prediction by pSORT (http://www.genscript.com/wolf-psort.html) |
b Localization prediction by TargetP 1.1 Server (http://www.cbs.dtu.dk/services/TargetP/) |
c Chloroplast localization signal confirmed by ChloroP (http://www.cbs.dtu.dk/services/ChloroP/) |
The CDS length of BnaGLYI members ranged from 324 bp (BnaGLYI34) to 3648 bp (BnaGLYI06), and the average CDS length is 785 bp. Accordingly, BnaGLYI06 encodes for the largest protein with a polypeptide length of 1216 aa and molecular weight of 134.9 kDa; while BnaGLYI34 encodes for the smallest protein with a polypeptide length of 108 aa and molecular weight of 12.2 kDa (Table 1). The isoelectric point (pI) of BnaGLYI proteins vary from 4.78 (BnaGLYI08) to 8.86 (BnaGLYI06). Most of the BnaGLYI members showed acidic pI value, and only four genes (BnaGLYI02, BnaGLYI05, BnaGLYI06 and BnaGLYI23) showed obvious alkalinity pI value (around 8.0) (Table 1). Moreover, there were also three members (BnaGLYI04, BnaGLYI19 and BnaGLYI31) showing basic pI value (Table 1). Sub-cellular localization of all identified BnaGLYI proteins were analyzed using three tools (CELLO, Wolf-pSORT and ChloroP). The results predicted that almost all BnaGLYI expect BnaGLYI15 were localized in cytoplasm. However, the Wolf-pSORT predicted that most of BnaGLYI were localized in the chloroplast, followed by the cytoplasm, nucleus, mitochondrion and cytoskeleton (Table 1).
Similarly, the CDS length of BnaGLYII genes also had very wide ranges, from 777 bp (BnaGLYII09) to 5745 bp (BnaGLYII24). Accordingly, BnaGLYII09 was the smallest protein (259 aa, 28.57 kDa); while BnaGLYII24 was the largest protein (1915 aa, 212.36 kDa) (Table 2). The pI value of BnaGLYII proteins varied from 5.21 (BnaGLYII29) to 9.04 (BnaGLYII04). Most of BnaGLYII proteins showed alkalinity with pI value around or more than 7.0, and only seven BnaGLYII proteins had an acidic pI value, including BnaGLYII09, BnaGLYII12, BnaGLYII13, BnaGLYII15, BnaGLYII17, BnaGLYII29 and BnaGLYII30 (Table 2). Sub-cellular locations of BnaGLYII proteins were different from BnaGLYI proteins. BnaGLYII proteins members were localized in different sub-cellular compartments, such as cytoplasm, chloroplast, extracellular matrix, outermembrane, periplasm, nucleus and mitochondrion (Table 2).
Table 2
Basic characteristics of BnaGLYI genes identified in Brassica napus L. genomes
Gene Symbol
|
Locus
Identifier
|
Location
|
Gene Start (bp)
|
Gene Stop (bp)
|
Strand
|
No. of Introns
|
CDS Length (bp)
|
PP Length (aa)
|
MW
(kDa)
|
pI
|
Localization
|
BnaGLYII01
|
BnaA01g03370D
|
A01
|
1597636
|
1600850
|
-
|
10
|
1056
|
352
|
39338.98
|
7.93
|
Cya; Chb, c
|
BnaGLYII02
|
BnaA02g25890D
|
A02
|
18962115
|
18965111
|
+
|
12
|
1848
|
616
|
68076.64
|
6.62
|
Cya, b
|
BnaGLYII03
|
BnaA03g14380D
|
A03
|
6611723
|
6613870
|
+
|
7
|
987
|
329
|
36085.29
|
8.81
|
Cya; Chb, c
|
BnaGLYII04
|
BnaA03g20230D
|
A03
|
9624333
|
9627034
|
-
|
8
|
1026
|
342
|
37496.84
|
9.04
|
Eca; Chb, c
|
BnaGLYII05
|
BnaA03g33050D
|
A03
|
15994860
|
15997418
|
+
|
10
|
1074
|
358
|
39988.96
|
7.17
|
Cya; Chb, c
|
BnaGLYII06
|
BnaA04g25070D
|
A04
|
18183840
|
18186578
|
-
|
7
|
993
|
331
|
36397.62
|
8.82
|
Eca; Chb, c
|
BnaGLYII07
|
BnaA05g03320D
|
A05
|
1854330
|
1860877
|
-
|
20
|
2868
|
956
|
106401.82
|
8.51
|
Oua; Chb, c
|
BnaGLYII08
|
BnaA05g11390D
|
A05
|
6376663
|
6378817
|
-
|
7
|
981
|
327
|
35883.92
|
8.07
|
Pea; Chb, c
|
BnaGLYII09
|
BnaA05g28080D
|
A05
|
20057585
|
20059255
|
+
|
6
|
777
|
259
|
28689.47
|
6.1
|
Pea; Cyb
|
BnaGLYII10
|
BnaA06g00850D
|
A06
|
603532
|
606586
|
+
|
8
|
1167
|
389
|
43530.78
|
7.74
|
Cya; Pea; Nub
|
BnaGLYII11
|
BnaA06g22690D
|
A06
|
15848234
|
15855367
|
+
|
23
|
3450
|
1150
|
126907.01
|
9.02
|
Cya; Chb, c
|
BnaGLYII12
|
BnaA08g01070D
|
A08
|
806559
|
808627
|
+
|
7
|
786
|
262
|
28574.49
|
6.19
|
Pea; Nub
|
BnaGLYII13
|
BnaA09g28480D
|
A09
|
21351908
|
21354940
|
-
|
11
|
1566
|
522
|
57465.81
|
6.19
|
Cya, b
|
BnaGLYII14
|
BnaA09g50050D
|
A09
|
33176404
|
33178430
|
+
|
7
|
969
|
323
|
35263.31
|
7.16
|
Cya; Chb, c
|
BnaGLYII15
|
BnaAnng27360D
|
Ann
|
31283476
|
31286304
|
-
|
4
|
2085
|
695
|
77448.79
|
5.81
|
Cya; Nub
|
BnaGLYII16
|
BnaC01g04650D
|
C01
|
2443496
|
2446457
|
-
|
10
|
1059
|
353
|
39822.41
|
6.96
|
Cya; Chb, c
|
BnaGLYII17
|
BnaC01g28980D
|
C01
|
27137154
|
27139952
|
-
|
4
|
2076
|
692
|
77157.53
|
5.81
|
Cya; Mtb
|
BnaGLYII18
|
BnaC02g47740D
|
C02
|
3827265
|
3830275
|
+
|
12
|
1848
|
616
|
68049.53
|
6.43
|
Cya, b
|
BnaGLYII19
|
BnaC03g17440D
|
C03
|
8920776
|
8922877
|
+
|
7
|
975
|
325
|
35687.84
|
8.85
|
Cya; Mtb; Chc
|
BnaGLYII20
|
BnaC03g24200D
|
C03
|
13557749
|
13560223
|
-
|
7
|
981
|
327
|
35840.97
|
8.85
|
Eca; Pea; Chb, c
|
BnaGLYII21
|
BnaC03g38140D
|
C03
|
23395036
|
23397633
|
+
|
10
|
1077
|
359
|
39993.97
|
7.14
|
Cya; Chb, c
|
BnaGLYII22
|
BnaC03g50930D
|
C03
|
35336691
|
35341520
|
-
|
16
|
2682
|
894
|
98725.64
|
8.7
|
Cya; Chb, c
|
BnaGLYII23
|
BnaC03g69760D
|
C03
|
59489758
|
59491865
|
-
|
7
|
870
|
290
|
31928.74
|
8.57
|
Cya; Chb, c
|
BnaGLYII24
|
BnaC04g02920D
|
C04
|
2066379
|
2078825
|
-
|
31
|
5745
|
1915
|
212357.58
|
8.24
|
Oua; Chb, c
|
BnaGLYII25
|
BnaC04g48930D
|
C04
|
47330538
|
47333344
|
-
|
7
|
996
|
332
|
36386.59
|
8.7
|
Eca; Chb, c
|
BnaGLYII26
|
BnaC05g20710D
|
C05
|
14195563
|
14198830
|
+
|
11
|
1566
|
522
|
57524.85
|
6.47
|
Cya, b
|
BnaGLYII27
|
BnaC06g06420D
|
C06
|
6895047
|
6896947
|
-
|
7
|
909
|
303
|
33196.01
|
7.21
|
Pea; Chb, c
|
BnaGLYII28
|
BnaC08g44630D
|
C08
|
37747241
|
37749209
|
-
|
7
|
984
|
328
|
35631.65
|
6.83
|
Pea; Chb, c
|
BnaGLYII29
|
BnaCnng30690D
|
Cnn
|
29167588
|
29174293
|
+
|
10
|
2448
|
816
|
93535.33
|
5.21
|
Cya, b
|
BnaGLYII30
|
BnaCnng46950D
|
Cnn
|
46379687
|
46381389
|
-
|
6
|
777
|
259
|
28653.35
|
5.72
|
Pea; Chb, c
|
Abbreviations: CDS: coding DNA sequence, PP: polypeptide length, MW: molecular weight, PI: isoelectric point, bp: base pair, aa: amino acid, kDa: kilodalton, Ch: chloroplast, Cy: cytosol, Mt: mitochondria, Nu: nucleus, Pe: periplasm, Ou: outermembrane, Ec: extracellular matrix. |
a Localization prediction by pSORT (http://www.genscript.com/wolf-psort.html) |
b Localization prediction by TargetP 1.1 Server (http://www.cbs.dtu.dk/services/TargetP/) |
c Chloroplast localization signal confirmed by ChloroP (http://www.cbs.dtu.dk/services/ChloroP/) |
Chromosome localization and gene duplication analysis of BnaGLYI and BnaGLYII
In order to get a more intuitive view of the distribution of GLY genes in B. napus chromosomes, the information of their starting and ending position in chromosomes were collected (Table 1), and chromosome distribution map was drawn on the MG2C (Map Gene2 Chromosome v2) website (Fig. 1). For BnaGLYI gene family, there were 31 genes were unevenly distributed on 14 chromosomes and no genes were found on chromosomes A01, A04, C01, C04 and C07. Four genes (BnaGLYI32-35) were located in the unmapped scaffolds in the Ann_random and Cnn_ random genome (Fig. 1a). Chromosomes A06, C05 and C08 contained the maximum number of BnaGLYI genes, four genes each; while five chromosomes (A02, A03, A05, C06 and C09) had only one BnaGLYI gene (Fig. 1a). Totally, 16 BnaGLYI genes distributed on the An-subgenome and 19 were from the Cn-subgenome. Similarly, it has been found that 27 BnaGLYII genes were unevenly located in 15 different chromosomes (Fig. 1b). There were four chromosomes that contained no BnaGLYII genes (A07, A10, C07 and C09) (Fig. 1b). Chromosome C03 harbored five BnaGLYII genes, which contained most BnaGLYI genes. However, most of chromosomes, like A01, A02, A04, A08, C02, C05, C06 and C08, contained only one BnaGLYII gene (Fig. 1b). The remaining three genes could not be mapped into chromosomes of the rapeseed genome, which were distributed on Ann and Cnn (Fig. 1b). Moreover, BnaGLYII proteins were mainly localized in the chloroplast and cytosol.
Gene replication is the main way of gene family expansion. This study also analyzed the gene replication of GLY gene family in B. napus. Among BnaGLYI proteins, there were 15 pairs duplicated genes. Three genes categorized into one group that formed three gene pairs (BnaGLYI01/17/18). Other gene pairs all included two genes (BnaGLYI02/19, BnaGLYI03/35, BnaGLYI04/22, BnaGLYI05/23, BnaGLYI07/25, BnaGLYI09/26, BnaGLYI10/28, BnaGLYI11/27, BnaGLYI12/32, BnaGLYI13/30, BnaGLYI14/29, BnaGLYI15/21). Moreover, it was found that nine gene pairs with over 95% sequence similarity in BnaGLYII proteins. They were BnaGLYII01/16, BnaGLYII02/18, BnaGLYII03/19, BnaGLYII04/20, BnaGLYII05/21, BnaGLYII06/24, BnaGLYII08/29, BnaGLYII11/22, BnaGLYII12/23, respectively (Fig. 1b). Such high level of sequence similarities implied the possibility of segmental duplication of those genes. And there were no tandem duplication found in both the BnaGLYI and BnaGLYII proteins.
Gene structure and phylogenetic analysis of the BnaGLYI and BnaGLYII
To further investigate the gene structure, the CDS and genomic sequences of the BnaGLY genes were aligned. It was shown that one to 26 introns varied from BnaGLYI genes and there were no introns in the BnaGLYI08 and BnaGLYI31 (Fig. 2, Table 1). BnaGLYI13, BnaGLYI15, BnaGLYI21 and BnaGLYI30 only contained one intron. BnaGLYI06 gene had the largest number of introns (26). Similarly, the BnaGLYII genes also contained varied numbers of introns (4–31); e.g., thirty-one introns were identified in BnaGLYII17, and 4 introns were predicted in BrGLYII24 (Fig. 2, Table 2). In addition, the GLY proteins that clustered in the same group possessed a similar structure.
To understand the evolutionary relationships and functions among the predicted BnaGLY proteins, a phylogenetic tree was drawn with all BnaGLY protein sequences by MEGA6.0. The 35 BnaGLY proteins were resolved into six distinguishing groups (group A-F; Fig. 3a). The largest clade Group F, containing 31 GLYI members, included 19 GLYI in Brassicaceae; whereas the smallest group (Clade B) containing 10 members, included 6 GLYI in Brassicaceae (Fig. 3a). AtGLYI4 and AtGLYI7 were in group F, whose transcription level could be induced by osmotic, extreme temperature and wounding [23]. BnaGLYI01 showed a high similarity to AtGLYI7. It was hypothesized that BnaGLYI01 may play similar roles in the response to stresses. Clade A included 15 members of Brassicaceae, one protein from Arabidopsis and one protein from rice. Group C included 27 proteins, which contained 12 members of Brassicaceae (six BnaGLYI, three BnaGLYI and three BolGLYI). Group D and E contained 12 and 14 members, respectively. Generally, BnaGLYI members first clustered with proteins from B. rapa and B. oleracea, followed by AtGLYIs, forming a small Brassicaceae clade, and subsequently further clustered with GLYI members from rice (Fig. 3a). It was reported that OsGly I-overexpressing rice line improved tolerance to NaCl, ZnCl2 and mannitol, and performed higher seed setting rate and yield compared to wild-type plants[31]. In Group F, BnaGLYI01, BnaGLYI17 and BnaGLYI18 that were highly similar to OsGly may have the similar roles in stress response.
Similarly, the BnaGLYII proteins formed eight distinct clades (Fig. 3b). Two BnaGLYII proteins were clustered in groups A, B, C, D, and F, respectively; four BnaGLYII proteins were classified in group E and G, and twelve BrGLYII proteins were in Group F (Fig. 3b).
Ka/Ks Ratio of BnaGLY genes
To explore the selective pressure on BnaGLY genes, we calculated the non-synonymous/synonymous mutation ratio (Ka/Ks); Ka/Ks > 1 indicates positive selection, Ka/Ks = 1 indicates neutral selection, and Ka/Ks < 1 indicates purifying selection[32]. The Ka/Ks ratio for 10 BnaGLYI genes was > 1, ranging from 1.0664 (BnaGLY11) to 6.3118 (BnaGLY102), which indicated that these genes were experienced higher positive selection pressure during the evolutionary history of B. napus. The other genes showed Ka/Ks < 1, which experienced higher purifying selection pressure (Table 3). Moreover, most of the BnaGLYII genes had a Ka/Ks < 1, only five BnaGLYII genes experienced positive selection pressure (Table 4). A comparison of Ka/Ks ratios among the BnaGLYI and BnaGLYII indicated that the average Ka/Ks ratio of BnaGLYI was higher than that of BnaGLYII genes. These results suggested that the BnaGLYI gene family may have suffered robust purifying selective pressure during evolution.
Table 3
Ka and Ks values for BnaGLYI paralogous gene pairs
No
|
Locus 1
|
Locus 2
|
Ks
|
Ka
|
Ka/Ks
|
Selection pressure
|
Average Ka/Ks
|
1
|
BnaGLYI11
|
BnaGLYI27
|
0.0482
|
0.0514
|
1.0664
|
Positive selection
|
|
2
|
BnaGLYI14
|
BnaGLYI29
|
0.0977
|
0.0384
|
0.3930
|
Purifying selection
|
|
3
|
BnaGLYI30
|
BnaGLYI15
|
0.036
|
0.1499
|
4.1639
|
Positive selection
|
|
4
|
BnaGLYI30
|
BnaGLYI21
|
0.0362
|
0.1173
|
3.2403
|
Positive selection
|
|
5
|
BnaGLYI32
|
BnaGLYI04
|
0.0744
|
0.1663
|
2.2352
|
Positive selection
|
|
6
|
BnaGLYI32
|
BnaGLYI22
|
0.0728
|
0.1182
|
1.6236
|
Positive selection
|
|
7
|
BnaGLYI03
|
BnaGLYI35
|
0.079
|
0.0134
|
0.1696
|
Purifying selection
|
|
8
|
BnaGLYI33
|
BnaGLYI05
|
0.1198
|
0.2261
|
1.8873
|
Positive selection
|
|
9
|
BnaGLYI33
|
BnaGLYI23
|
0.1184
|
0.2442
|
2.0625
|
Purifying selection
|
1.8559
|
10
|
BnaGLYI19
|
BnaGLYI02
|
0.0418
|
0.013
|
0.3110
|
Purifying selection
|
|
11
|
BnaGLYI07
|
BnaGLYI10
|
0.2567
|
0.1386
|
0.5399
|
Purifying selection
|
|
12
|
BnaGLYI07
|
BnaGLYI28
|
0.2567
|
0.1236
|
0.48150
|
Purifying selection
|
|
13
|
BnaGLYI25
|
BnaGLYI10
|
0.3206
|
0.1386
|
0.4323
|
Purifying selection
|
|
14
|
BnaGLYI25
|
BnaGLYI28
|
1.3206
|
0.1236
|
0.0936
|
Purifying selection
|
|
15
|
BnaGLYI23
|
BnaGLYI05
|
0.0928
|
0.066
|
0.7112
|
Purifying selection
|
|
16
|
BnaGLYI22
|
BnaGLYI12
|
0.0364
|
0.2007
|
5.5137
|
Positive selection
|
|
17
|
BnaGLYI10
|
BnaGLYI28
|
0.0448
|
0.0128
|
0.2857
|
Purifying selection
|
|
18
|
BnaGLYI13
|
BnaGLYI15
|
0.0358
|
0.1339
|
3.7402
|
Positive selection
|
|
19
|
BnaGLYI12
|
BnaGLYI04
|
0.0372
|
0.2348
|
6.3118
|
Positive selection
|
|
Table 4
Ka and Ks values for BnaGLYII paralogous gene pairs
No
|
Locus 1
|
Locus 2
|
Ks
|
Ka
|
Ka/Ks
|
Selection pressure
|
Average Ka/Ks
|
1
|
BnaGLYII09
|
BnaGLYII30
|
0.12470
|
0.05000
|
0.4010
|
Purifying selection
|
|
2
|
BnaGLYII24
|
BnaGLYII04
|
0.05800
|
0.06350
|
1.0948
|
Positive selection
|
|
3
|
BnaGLYII24
|
BnaGLYII07
|
0.02070
|
0.02720
|
1.3140
|
Positive selection
|
|
4
|
BnaGLYII29
|
BnaGLYII03
|
0.11980
|
0.08840
|
0.7379
|
Purifying selection
|
|
5
|
BnaGLYII29
|
BnaGLYII08
|
0.03070
|
0.03670
|
1.1954
|
Positive selection
|
|
6
|
BnaGLYII25
|
BnaGLYII06
|
0.11250
|
0.03930
|
0.3493
|
Purifying selection
|
|
7
|
BnaGLYII19
|
BnaGLYII08
|
0.12060
|
0.07830
|
0.6493
|
Purifying selection
|
|
8
|
BnaGLYII19
|
BnaGLYII03
|
0.04170
|
0.02420
|
0.5803
|
Purifying selection
|
|
9
|
BnaGLYII10
|
BnaGLYII27
|
0.08610
|
0.03340
|
0.3879
|
Purifying selection
|
|
10
|
BnaGLYII10
|
BnaGLYII12
|
0.19390
|
0.10200
|
0.5260
|
Purifying selection
|
|
11
|
BnaGLYII02
|
BnaGLYII18
|
0.01030
|
0.00890
|
0.8641
|
Purifying selection
|
|
12
|
BnaGLYII28
|
BnaGLYII14
|
0.07520
|
0.05860
|
0.7793
|
Purifying selection
|
0.7978
|
13
|
BnaGLYII23
|
BnaGLYII12
|
0.05060
|
0.03380
|
0.6680
|
Purifying selection
|
|
14
|
BnaGLYII08
|
BnaGLYII03
|
0.13160
|
0.07190
|
0.5464
|
Purifying selection
|
|
15
|
BnaGLYII11
|
BnaGLYII22
|
0.04280
|
0.03320
|
0.7757
|
Purifying selection
|
|
16
|
BnaGLYII15
|
BnaGLYII17
|
0.05250
|
0.01200
|
0.2286
|
Purifying selection
|
|
17
|
BnaGLYII13
|
BnaGLYII26
|
0.06540
|
0.04560
|
0.6973
|
Purifying selection
|
|
18
|
BnaGLYII20
|
BnaGLYII04
|
0.01020
|
0.03350
|
3.2843
|
Positive selection
|
|
19
|
BnaGLYII20
|
BnaGLYII24
|
0.06890
|
0.07010
|
1.0174
|
Positive selection
|
|
20
|
BnaGLYII27
|
BnaGLYII12
|
0.18670
|
0.08020
|
0.4296
|
Purifying selection
|
|
21
|
BnaGLYII01
|
BnaGLYII16
|
0.06060
|
0.04020
|
0.6634
|
Purifying selection
|
|
22
|
BnaGLYII21
|
BnaGLYII05
|
0.03470
|
0.00580
|
0.1672
|
Purifying selection
|
|
23
|
BnaGLYII07
|
BnaGLYII04
|
0.08040
|
0.07980
|
0.9925
|
Purifying selection
|
|
Conserved motif and the enzyme activity of BnaGLY
Based on the conserved metal binding sites H/QEH/QE of GLYI proteins and multiple alignment results, the expected enzyme activity of the putative BnaGLYI proteins was predicted. Overall, out of a total 35 putative BnaGLYI proteins, sixteen have all the four conserved residues and are expected to have functional GLYI enzyme activity (Fig. 4 and Table 5). The other 19 BnaGLYI proteins may have variant function. Additionally, GLYII from microorganism and plant contained the well conserved metal binding motif (THXHXDH) and active site motif (C/GHT) [26, 33], which play an important role in the GLYII enzyme activity. Therefore, based on protein sequence alignment results, the presence of enzymatic activity of the putative BnaGLYII proteins were evaluated (Fig. 5, Table 6). Fourteen of 30 putative BnaGLYII proteins possess all the conserved metal binding residues and may have the functional GLYII enzyme activity (Table 6).
Table 5
Analysis of the putative BnaGLYI for their activity and metal ion dependency
Proteins
|
Metal binding sites Expected GLYI
|
Expected GLYI
enzyme activity
|
Length of GLYI
domain (aa)
|
H/Q
|
E
|
H/Q
|
E
|
BnaGLYI1
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI2
|
√
|
—
|
√
|
√
|
No
|
|
BnaGLYI3
|
√
|
—
|
√
|
√
|
No
|
|
BnaGLYI4
|
—
|
√
|
—
|
—
|
No
|
|
BnaGLYI5
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI6
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI7
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI8
|
√
|
—
|
√
|
√
|
No
|
|
BnaGLYI9
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI10
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI11
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI12
|
—
|
√
|
—
|
—
|
No
|
|
BnaGLYI13
|
√
|
—
|
—
|
—
|
No
|
|
BnaGLYI14
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI15
|
√
|
—
|
—
|
—
|
No
|
|
BnaGLYI16
|
√
|
—
|
√
|
√
|
No
|
|
BnaGLYI17
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI18
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI19
|
√
|
—
|
√
|
√
|
No
|
|
BnaGLYI20
|
—
|
√
|
—
|
√
|
No
|
|
BnaGLYI21
|
√
|
—
|
—
|
—
|
No
|
|
BnaGLYI22
|
—
|
√
|
—
|
—
|
No
|
|
BnaGLYI23
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI24
|
√
|
—
|
√
|
√
|
No
|
|
BnaGLYI25
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI26
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI27
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI28
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI29
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI30
|
√
|
—
|
—
|
—
|
No
|
|
BnaGLYI31
|
√
|
—
|
√
|
—
|
No
|
|
BnaGLYI32
|
—
|
√
|
—
|
—
|
No
|
|
BnaGLYI33
|
—
|
√
|
√
|
√
|
No
|
|
BnaGLYI34
|
—
|
—
|
—
|
—
|
No
|
|
BnaGLYI35
|
√
|
—
|
√
|
√
|
No
|
|
BnaGLYI6
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI7
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI8
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI9
|
√
|
√
|
√
|
√
|
Yes
|
|
BnaGLYI10
|
√
|
√
|
√
|
√
|
Yes
|
|
Table 6
Analyses of the putative BnaGLYII proteins for their activity and conserved motifs
Proteins
|
Conserved metal binding motif (THHHXDH)
|
Active site motif (C/GHT)
|
Proteins Expected GLYII enzyme activity
|
BnaGLYII1
|
—
|
√
|
No
|
BnaGLYII2
|
—
|
—
|
No
|
BnaGLYII3
|
√
|
√
|
Yes
|
BnaGLYII4
|
√
|
√
|
Yes
|
BnaGLYII5
|
—
|
—
|
No
|
BnaGLYII6
|
√
|
—
|
No
|
BnaGLYII7
|
√
|
√
|
Yes
|
BnaGLYII8
|
√
|
√
|
Yes
|
BnaGLYII9
|
—
|
√
|
No
|
BnaGLYII10
|
—
|
√
|
No
|
BnaGLYII11
|
—
|
—
|
No
|
BnaGLYII12
|
—
|
√
|
No
|
BnaGLYII13
|
√
|
√
|
Yes
|
BnaGLYII14
|
√
|
√
|
Yes
|
BnaGLYII15
|
—
|
—
|
No
|
BnaGLYII16
|
—
|
√
|
No
|
BnaGLYII17
|
—
|
—
|
No
|
BnaGLYII18
|
—
|
—
|
No
|
BnaGLYII19
|
√
|
√
|
Yes
|
BnaGLYII20
|
√
|
√
|
Yes
|
BnaGLYII21
|
—
|
—
|
No
|
BnaGLYII22
|
—
|
—
|
No
|
BnaGLYII23
|
—
|
√
|
No
|
BnaGLYII24
|
√
|
√
|
Yes
|
BnaGLYII25
|
√
|
√
|
Yes
|
BnaGLYII26
|
√
|
√
|
Yes
|
BnaGLYII27
|
—
|
√
|
No
|
BnaGLYII28
|
√
|
√
|
Yes
|
BnaGLYII29
|
√
|
√
|
Yes
|
BnaGLYII30
|
√
|
√
|
Yes
|
To better understand the protein sequence features of BnaGLY, the conserved motifs of each protein were also identified using MEME program (http://meme-suite.org/tools/meme) (Additional file 1 : Fig. S1). We found that most proteins in the same group had similar motifs, and the LOGOs of these protein motifs were obtained by MEME (Additional file 2 : Fig. S2). In BnaGLYI proteins, motifs 1 were observed in all the 34 BnaGLYI proteins except the BnaGLYI20. Motif 1, 2, 3 and 4 contained the conserved sites of H/QEH/QE. Similarly, motifs 1 were detected in most BnaGLYII proteins except the BnaGLY16 and BnaGLY26. Motif 1 and 3 contain the metal binding site (THXHXDH) and active site (C/GHT), respectively. Moreover, we also found that most proteins in the same group had similar motifs (Fig. S2).
Tissue expression profiles of BnaGLYI and BnaGLYII
To investigate the tissue-specific expression profiles of the BnaGLY genes, we selected 20 types of tissues in database (https://biodb.swu.edu.cn/brassica/) to analysis, including 10 tissues at the initial flowering stage and 10 tissues at the different development stages of the podding. The results indicated that the expression levels of the BnaGLY genes showed diverse expression patterns (Fig. 6). Six BnaGLYI members (BnaGLYI09, BnaGLYI11, BnaGLYI16, BnaGLYI26, BnaGLYI27 and BnaGLYI35) were highly expressed in most of the tissues. Their average fragments per kilo base of exon sper million fragments mapped (FPKM) values were over 25 and the value in single tissue was at leastover 3. In contrast, the expression levels of BnaGLYI22, BnaGLYI29, BnaGLYI32 and BnaGLYI34 were not detected in most tissues. They only showed faint expression in one to three tissues, e.g., the expression of BnaGLYI32 was only detected in silique pericarps (SP) at 24 and 27 days after flowering (DAF). The expression of BnaGLYI14 was not detected in all the 10 tissues (Fig. 6a). In BnaGLYII genes, BnaGLYII28 were only weakly expressed in SP at 27 DAF, while the FPKM values of BnaGLYII18 and BnaGLYII29 were Less than 1. The other BnaGLYII genes were constitutive expression. However, some genes were expressed at higher levels in specific tissues, e.g., BnaGLYII19 were highly expressed in anther (Fig. 6b). These results indicated that most BnaGLY genes were expressed at different levels in different tissues.
Moreover, the expression levels of BnaGLY in developing seeds at two, four, six and eight weeks after pollination (WAP) were analyzed using the gene expression omnibus (GEO) database (GSE77637) (Fig. 7). The expression of BnaGLYI11 and BnaGLYI27 gene was high in the four stages and the FPKM values were all over 2000. The BnaGLYI01, BnaGLYI04, and BnaGLYI32 showed high expression level at eight WAP. BnaGLYI10, BnaGLYI14, BnaGLYI28, BnaGLYI29 and BnaGLYI34 showed low expression levels in all the seed development stages (Fig. 7a). In the case of BnaGLYII, the expression of BnaGLYII09, BnaGLYII14 and BnaGLYII28 was upregulated at eight WAP. The expression of BnaGLYII03, BnaGLYII06, BnaGLYII24, BnaGLYII13, BnaGLYII25, BnaGLYII19, BnaGLYII20 and BnaGLYII26 were gradually decreased in the four development stages (Fig. 7b).
The expression of the BnaGLY during seed germination
To dissect the influence of GLY pathways in seed germination of B. napus, the expression level of GLY genes was analyzed using the data in GEO (GSE13723). The expression level of BnaGLYI in winter oilseed rape (WOSR) accessions with different germination rates (high, medium and low; C129, C033 and C032, respectively) at different germination times showed similar expression pattern, e.g., the expression of BnaGLYI11 and BnaGLYI27 were significantly up-regulated at 72 hour after imbibition (hai) in all the three accessions, and the expression of BnaGLYI04, BnaGLYI07, BnaGLYI17, BnaGLYI21 and BnaGLYI25 obviously decreased at 36 hai and 72 hai. Meanwhile, the BnaGLYI06, BnaGLYI10, BnaGLYI14, BnaGLYI24, BnaGLYI28, BnaGLYI29 and BnaGLYI34 showed weak expression in all the accessions at the four germination stages (Fig. 8a). In contrast, the average expression level of BnaGLYII genes was much higher than that of BnaGLYI. BnaGLYII5, BnaGLYII11, BnaGLYII21 and BnaGLYII22 showed significantly up-regulation at 36 hai and 72 hai in all the three accessions; while BnaGLYII1 were only up-regulated at 72 hai in the three accessions. BnaGLYII9 showed high expression at early stage of germination, however, sharply was decreased at 36 hai, and then was significantly up-regulated at 72 hai. BnaGLYII3 and BnaGLYII19 were up-regulated at the three germination time points in the three accessions. BnaGLYII17 and BnaGLYII25 showed very low expression levels in accession C033 from 0 to 72 hai (Fig. 8b). The results demonstrated that BnaGLY showed diverse expression patterns, though some genes were clustered within the same group.
The response of the BnaGLYI and BnaGLYII on pathogen infection
The RNA-seq data sets available in GEO (GSE81545) were used for analysis the BnaGLY gene expression in response to the fungal pathogen [34]. In BnaGLYI gene, the expression level of BnaGLYI07, BnaGLYI24 and BnaGLYI25 were significantly up-regulated after S. sclerotiorum pathogen infection in the resistant cultivar (ZY821) and sensitive cultivar (Westar). BnaGLYI02, BnaGLYI19, BnaGLYI31 and BnaGLYI35 were significantly down-regulated after pathogen infection in both cultivars. While the expression of BnaGLYI08 and BnaGLYI10 were down–regulated in sensitive cultivar; however, their expression were up–regulated in resistant cultivar. Most interestingly, the BnaGLYI17 and BnaGLYI18 gene were up-regulated in the susceptible cultivar; however, they were down-regulated in the resistant cultivar (Fig. 9a).
Different from the BnaGLYII genes, most BnaGLYII genes showed significantly decreased expression levels after pathogen infection, e.g., BnaGLYII06, BnaGLYII08, BnaGLYII14, BnaGLYII23, BnaGLYII25, BnaGLYII28 and BnaGLYII30 were significantly down-regulated in both cultivars. Only BnaGLYII02, BnaGLYII03, BnaGLYII18 and BnaGLYII19 were significantly induced in both cultivars after infected by pathogen (Fig. 9b). Moreover, BnaGLYII16 was up-regulated in the susceptible cultivars, and down-regulated in the resistant cultivars.