RNA sequencing and analysis of differentially expressed genes
The transcriptome changes of BA pulp were investigated through RNA-Seq analysis. More than 40 million reads were generated per sample. Of these reads, the Q30 percentage (sequencing error rate < 1%) was over 93%, and GC content was approximately 47% for the libraries. Among all the libraries, 64.85–66.32% of unique reads were mapped to the Pyrus bretschneideri Rehd (Table 1).
Table 1
Statistics of sequencing data of the six libraries.
|
Library
|
Clean reads
|
Q30 ratio (%)
|
GC content (%)
|
Mapping ratio (%)
|
BA1
|
42034166
|
93.59
|
46.93
|
27270180(64.88%)
|
BA2
|
44906822
|
93.64
|
46.91
|
29120166(64.85%)
|
BA3
|
41265050
|
93.64
|
47.02
|
27011504(65.46%)
|
CK1
|
45391358
|
93.73
|
47.11
|
30102908(66.32%)
|
CK2
|
43646738
|
93.86
|
46.82
|
28547478(65.41%)
|
CK3
|
42463764
|
93.6
|
46.67
|
27619994(65.04%)
|
A total of 3146 genes and 1145 genes in BA pulp were significantly up-regulated and down-regulated, respectively (Fig. 1A). All the DEGs were subjected to GO and KEGG analysis. In the GO database, thylakoid, thylakoid part, photosynthetic membrane, photosystem, et al were the most enriched in the ‘cellular component’ category; iron ion binding, DNA binding transcription factor activity, et al were the most enriched in the ‘molecular function’ category cellular component (Fig. 1B). Pathways showing significant change (Q value ≤ 0.05) in BA pulp were identified using the KEGG database. The DEGs were mainly involved in the pathways of plant hormone signal transduction, starch and sucrose metabolism, photosynthesis, phenylpropanoid biosynthesis, flavonoid biosynthesis, carotenoid biosynthesis, brassinosteroid biosynthesis and et al (Fig. 1 C).
Changes of sugar metabolism-related gene expression in BA fruit
Sorbitol metabolism related gene analysis showed that a sorbitol synthesis gene, S6PDH (LOC103966946), was significantly down-regulated. The expression levels of three sorbitol dehydrogenase genes (LOC103960512, LOC103930819 and LOC103930934), seven sorbitol transporter genes (LOC103936915, LOC103964420, LOC103948508, LOC103929477, LOC103936910, LOC103936324 and LOC103964421) and a starch degradation gene (LOC103934256) were significantly increased in BA fruit. Four sucrose-phosphate synthase genes (LOC103946723, LOC103937917, LOC103952486 and LOC103956574) and two sucrose synthase genes (LOC103927263 and LOC103946870) were significantly down-regulated and up-regulated after the treatment of boric acid, respectively (Table 2).
Table 2
List of DEGs relate to sorbitol, sucrose and starch metabolism
|
ID
|
Gene description
|
log2Fold Change
|
Sorbitol metabolism
|
|
|
LOC103966946
|
D-sorbitol-6-phosphate dehydrogenase, S6PDH
|
-1.59
|
LOC103960512
|
Sorbitol dehydrogenase, SDH
|
1.34
|
LOC103930819
|
Sorbitol dehydrogenase, SDH
|
1.46
|
LOC103930934
|
Sorbitol dehydrogenase, SDH
|
8.02
|
LOC103936915
|
Sorbitol transporter, SOT
|
4.67
|
LOC103964420
|
Sorbitol transporter, SOT
|
4.28
|
LOC103948508
|
Sorbitol transporter, SOT
|
5.99
|
LOC103929477
|
Sorbitol transporter, SOT
|
8.85
|
LOC103936910
|
Sorbitol transporter, SOT
|
6.96
|
LOC103936324
|
Sorbitol transporter, SOT
|
5.73
|
LOC103964421
|
Sorbitol transporter, SOT
|
4.65
|
Sucrose metabolism
|
|
|
LOC103946723
|
Sucrose-phosphate synthase, SPS
|
-2.29
|
LOC103937917
|
Sucrose-phosphate synthase, SPS
|
-2.55
|
LOC103952486
|
Sucrose-phosphate synthase, SPS
|
-3.80
|
LOC103956574
|
Sucrose-phosphate synthase, SPS
|
-2.54
|
LOC103927263
|
Sucrose synthase, SS
|
1.25
|
LOC103946870
|
Sucrose synthase, SS
|
3.26
|
Starch degradation
|
|
|
LOC103934256
|
β-amylase
|
3.08
|
Changes of fatty acid synthesis-related gene expression in BA fruit
Six genes involved in fatty acid biosynthesis were significantly regulated induced by BA. These genes included two malonyl CoA-acyl carrier protein transacylase genes (LOC103945733 and LOC103935263), a 3-oxoacyl-[acyl-carrier-protein] synthase gene (LOC103951262), a 3-hydroxyacyl-[acyl-carrier-protein] dehydratase gene (LOC103967963) and three enoyl-[acyl-carrier-protein] reductase genes (LOC103964860, LOC103959004 and LOC103940555) (Table 3).
Table 3
List of DEGs relate to fatty acid biosynthesis
|
ID
|
Gene description
|
log2Fold Change
|
Fatty acid biosynthesis
|
|
|
LOC103945733
|
Malonyl CoA-acyl carrier protein transacylase
|
1.50
|
LOC103935263
|
Malonyl CoA-acyl carrier protein transacylase
|
1.35
|
LOC103951262
|
3-oxoacyl-[acyl-carrier-protein] synthase
|
1.23
|
LOC103967963
|
3-hydroxyacyl-[acyl-carrier-protein] dehydratase
|
1.40
|
LOC103964860
|
Enoyl-[acyl-carrier-protein] reductase
|
1.83
|
LOC103959004
|
Enoyl-[acyl-carrier-protein] reductase
|
2.43
|
LOC103940555
|
Enoyl-[acyl-carrier-protein] reductase
|
1.61
|
Changes of plant hormone metabolism-related gene expression in BA fruit
Analysis of DEGs found that two indole-3-acetaldehyde oxidase genes (LOC103958137 and LOC103929079) and six Peroxidase genes (LOC103948174, LOC103951047, LOC103934752, LOC103964015, LOC103958862 and LOC103953577) which involved in IAA synthesis and degradation, respectively, were significantly down-regulated and up-regulated in BA fruit. Moreover, the expression levels of two GA synthesis genes (LOC108865311, LOC103948777 and LOC103939254) were significantly increased in BA fruit. Four key genes of ethylene synthesis (LOC103948231, LOC103943975 and LOC103939367) were significantly decreased in BA fruit (Table 4).
Table 4
List of DEGs relate to plant hormone metabolism
|
ID
|
Gene description
|
log2Fold Change
|
IAA degradation
|
|
|
LOC103958137
|
indole-3-acetaldehyde oxidase
|
-4.93
|
LOC103929079
|
indole-3-acetaldehyde oxidase
|
-4.50
|
LOC103948174
|
Peroxidase, POD
|
1.60
|
LOC103951047
|
Peroxidase, POD
|
6.54
|
LOC103934752
|
Peroxidase, POD
|
2.43
|
LOC103964015
|
Peroxidase, POD
|
3.11
|
LOC103958862
|
Peroxidase, POD
|
1.80
|
LOC103953577
|
Peroxidase, POD
|
6.48
|
GA synthesis
|
|
|
LOC108865311
|
ent-kaurene oxidase, KO
|
3.90
|
LOC103948777
|
ent-kaurenoic acid oxidase, KAO
|
1.81
|
LOC103939254
|
ent-kaurenoic acid oxidase, KAO
|
3.83
|
Ethylene synthesis
|
|
|
LOC103948231
|
1-aminocyclopropane-1-carboxylate oxidase, ACO
|
-7.01
|
LOC103943975
|
1-aminocyclopropane-1-carboxylate oxidase, ACO
|
-9.88
|
LOC103939367
|
1-aminocyclopropane-1-carboxylate oxidase, ACO
|
-1.74
|
LOC103956316
|
1-aminocyclopropane-1-carboxylate synthase, ACS
|
-2.39
|