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Acta Agronomica Sinica ›› 2026, Vol. 52 ›› Issue (10): 2912-2926.doi: 10.3724/SP.J.1006.2026.65001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Comparative transcriptome analysis reveals the molecular mechanisms underlying differences in differentiation capacity between two distinct callus types in Brassica napus

Yang Qin-Li1(), Zhang Xiao-Ling1, Zhang Li-Xian1,2, Li Hong-Li1,2, Zhang Huan-Yang1, Li Huan-Li1, Sun Cai-Hong1,2, Li Jing1, Zhu Yong-Hong1, Sun Xuan1, Yao Lin1, Wang Dan1, Shang-Guan Xiao-Xia1,*()   

  1. 1 Institute of Cotton Research, Shanxi Agricultural University, Yuncheng 044000, Shanxi, China
    2 College of Agriculture, Shanxi Agricultural University, Jinzhong 030800, Shanxi, China
  • Received:2026-01-01 Accepted:2026-07-15 Online:2026-10-12 Published:2026-07-21
  • Contact: Shang-Guan Xiao-Xia, E-mail: sgxx74@126.com
  • Supported by:
    ‘Unveiling the List and Taking Command’ Project of Shanxi Province Science and Technology Major Special Project(202201140601025-5-04);Yuncheng City Science and Technology Plan Project(YCKJ-2025046)

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Abstract:

The genetic transformation efficiency of Brassica napus is severely constrained by callus differentiation capacity, which varies greatly among genotypes. In this study, transcriptome analysis was performed at two key time points to elucidate the molecular mechanisms underlying differences in callus differentiation capacity, thereby providing a theoretical basis and genetic resources for genetic improvement. Two representative materials with highly significant differences in callus differentiation capacity, ZP10 with high differentiation capacity and ZP15 with low differentiation capacity, were selected from 20 B. napus genotypes. Transcriptome sequencing and comparative analysis were conducted on calli from these two lines at 0 and 30 days after induction (DAI). A total of 1915 genes were identified through differentially expressed gene (DEG) screening. GO and KEGG enrichment analyses showed that these genes were significantly enriched in pathways related to plant hormone signal transduction, photosynthesis-antenna proteins, photosynthetic organ assembly, and cell wall biosynthesis. Combined with weighted gene co-expression network analysis (WGCNA), 25 key candidate genes were ultimately identified, and their expression patterns were closely associated with high differentiation capacity. The reliability of the transcriptome data was confirmed by qRT-PCR. In conclusion, the high callus differentiation capacity of B. napus is associated with a complex regulatory network coordinated by multiple genes and involving hormone signaling responses, initial establishment of the photosynthetic system, and cell wall remodeling. The key genes identified in this study provide important targets for further elucidating the molecular mechanisms underlying rapeseed regeneration and for breeding genotypes with high transformation efficiency.

Key words: Brassica napus L., genetic transformation, callus, differentiation ability, RNA-Seq

Table 1

Primer sequences"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)
BnActin-F TGTGCCAATCTACGAGGGTTTC
BnActin-R TCTCACAATTTCCCGCTCGG
BnaC07G0469000ZS-F (ABI1-F) GACAACAACGGCGAGACTTC
BnaC07G0469000ZS-R (ABI1-R) TCCTCTCTCTACAATACTCCGCA
BnaA07G0340700ZS-F (EIL3-F) TCAGGCAACGAAGGAGGGGC
BnaA07G0340700ZS-R (EIL3-R) AGGAGGACTCTGGTTCTGCGGC
BnaA08G0267000ZS-F (CAP10B-F) GTGCTCAGTGGTCTTGGTCC
BnaA08G0267000ZS-R (CAP10B-R) GAGAAGGGACCCGAACGAG
BnaC02G0368500ZS-F (LHCB5-F) CACGCCAGATGGGCTATG
BnaC02G0368500ZS-R (LHCB5-R) AGAACAACCTCAGCAACTACAGC
BnaC04G0266800ZS-F (LHCA2-F) CCTGGTGACTTCGGGTTTGAT
BnaC04G0266800ZS-R (LHCA2-R) TTGTTGTTTGGGAAGATTGGGTC
BnaC04G0553100ZS-F (PAL1-F) TGTGAAAGCGAGTAGTGATTGGG
BnaC04G0553100ZS-R (PAL1-R) CTCTTGTGGCGGAGTGTGGTA

Table 2

Callus induction and differentiation of different rapeseed lines"

材料编号
Material number		 接种外植体数
Number of inoculated explants		 愈伤组织的诱导率
Callus induction rate (%)		 出愈伤后接种外植体数
Number of explants
inoculated post-callus		 分化率
Callus differentiation
rate (%)		 再生苗率
Plant regeneration rate (%)
ZP1 60 47.78 def 30 47.22 bcdef 23.33 def
ZP2 60 43.33 defg 30 43.89 bcdefg 22.22 ef
ZP3 60 41.67 efg 30 40.00 defgh 20.00 fg
ZP4 60 55.00 cde 30 56.67 b 28.89 cde
ZP5 60 36.11 fg 30 36.11 efgh 18.89 fg
ZP6 60 41.67 efg 30 42.78 bcdefg 22.22 ef
ZP7 60 70.56 ab 30 72.22 a 37.78 b
ZP8 60 31.11 gh 30 32.22 gh 18.89 fg
ZP9 60 52.78 cde 30 55.56 bc 31.11 bcd
ZP10 60 80.56 a 30 80.00 a 46.67 a
ZP11 60 45.56 def 30 46.11 bcdefg 25.56 cdef
ZP12 60 53.89 cde 30 48.33 bcde 25.56 cdef
ZP13 60 21.67 h 30 38.89 efgh 13.33 g
ZP14 60 41.67 efg 30 41.67 cdefgh 24.44 cdef
ZP15 60 63.89 bc 30 27.78 h 20.00 fg
ZP16 60 47.22 def 30 45.00 bcdefg 22.22 ef
ZP17 60 34.44 fg 30 33.89 fgh 32.22 bc
ZP18 60 70.00 ab 30 69.44 a 36.67 b
ZP19 60 56.11 cd 30 53.89 bcd 26.67 cdef
ZP20 60 55.00 cde 30 56.11 b 26.67 cdef

Fig. 1

Callus morphology of ZP10 and ZP15 at different differentiation stages Scale bars: 1 cm for the overall morphology of calli in petri dishes and 1 mm for callus micrographs. DAI: days after induction."

Table 3

RNA sequencing data output and quality control metrics"

样本
Sample		 原始读数
Raw reads		 清洁读数
Clean reads		 清洁读段占比
Proportion of clean reads (%)		 Q20
(%)		 Q30
(%)		 平均GC含量
Mean GC content
(%)
YS10-1 44,413,342 43,995,642 99.06 98.87 96.46 46.72
YS10-2 39,924,584 39,504,914 98.95 98.81 96.31 46.67
YS10-3 43,630,348 43,304,644 99.25 98.84 96.42 46.80
YS15-1 48,312,106 48,011,976 99.38 98.97 96.78 46.88
YS15-2 43,486,404 43,233,720 99.42 98.85 96.51 46.73
YS15-3 43,992,790 43,732,868 99.41 98.89 96.61 46.81
FH10-1 41,487,832 41,156,180 99.20 98.77 96.25 46.78
FH10-2 43,749,892 43,398,526 99.20 98.83 96.42 46.87
FH10-3 39,741,766 39,332,990 98.97 98.91 96.65 46.94
FH15-1 41,207,924 40,916,276 99.29 98.86 96.48 46.98
FH15-2 40,900,106 40,570,336 99.19 98.76 96.13 46.98
FH15-3 39,623,008 39,298,456 99.18 98.87 96.47 47.04

Fig. 2

Visualization of inter-sample correlations based on transcriptome data A: PCA scatter plot; B: inter-sample correlation heatmap. Abbreviations are the same as those given in Table 3."

Fig. 3

Analysis of differentially expressed genes in ZP10 and ZP15 at different stages A: grouped volcano plot of differentially expressed genes; B: clustered heatmap of DEGs. Abbreviations are the same as those given in Table 3."

Fig. 4

Integrated analysis of DEG overlap and expression patterns A: venn diagram of DEGs; B: cluster analysis of differentially expressed genes. Abbreviations are the same as those given in Table 3."

Table 4

Significantly upregulated transcription factor families and representative members in ZP10 calli"

转录因子
家族
TF family		 上调的转录
因子数量
Number of up-regulated TFs		 代表性成员
Representative
member		 拟南芥同源
基因
Arabidopsis ortholog		 上调倍数
Fold change		 功能预测
Putative function
AP2/ERF 14 BnaC09G0490600ZS BBM1 9.40 参与锌离子或其他金属/小分子的跨膜运输过程
Involved in the transmembrane transport of zinc ions, other metal ions, or small molecules
ARR-B 2 BnaC09G0597600ZS gluA 10.39 一种定位于溶酶体的糖基水解酶
A lysosome‑localized glycosyl hydrolase
NAC 5 BnaC02G0167800ZS At1g02270 7.48 参与钙信号调控的核酸代谢或磷酸化过程
Involved in nucleic acid metabolism or phosphorylation processes regulated by calcium signaling
LBD 4 BnaC09G0523600ZS ZIFL1 8.65 在生长素运输和干旱胁迫响应中发挥关键作用
Play key roles in auxin transport and drought stress response
WRKY 7 BnaC02G0135100ZS EXO70B1 9.80 在囊泡运输和免疫信号传导中起作用
Play roles in vesicle transport and immune signaling
bHLH 13 BnaC09G0549300ZS MBR1 8.01 与MBR2协同作用, 调控茉莉酸信号通路
Acts synergistically with MBR2 to regulate the jasmonic acid signaling pathway
MYB 3 BnaA02G0407800ZS EXL2 12.02 在连接营养信号与植物生长调控中发挥作用
Involved in linking nutrient signals to plant growth regulation
bZIP 5 BnaC02G0155400ZS HSP90-2 8.07 参与植物生长发育、环境信号整合及免疫应答等
Involved in plant growth and development, environmental signal integration, and immune responses

Fig. 5

GO enrichment analysis of differentially expressed genes during callus differentiation"

Fig. 6

KEGG enrichment analysis of differentially expressed genes during callus differentiation The degree of enrichment increases with the enrichment score; dot size indicates the number of enriched genes, and darker red indicates higher enrichment significance."

Fig. 7

Heatmap of correlations between samples and modules Abbreviations are the same as those given in Table 3. In each module, the upper number indicates the correlation coefficient r, and the number in parentheses below indicates the P-value."

Fig. 8

Expression heatmap of candidate genes in different materials Abbreviations are the same as those given in Table 3. Colors from blue to red indicate low to high relative gene expression levels, expressed as Z-scores."

Fig. 9

qRT-PCR validation of differentially expressed genes Abbreviations are the same as those given in Table 3. Data are presented as mean ± standard deviation (SD) (n = 3). Different lowercase letters above the bars indicate significant differences at P < 0.05."

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