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作物学报

作物学报 ›› 2025, Vol. 51 ›› Issue (11): 3105-3118.doi: 10.3724/SP.J.1006.2025.55026

• 研究简报 • 上一篇    

甘蓝型油菜响应碱胁迫的基因表达差异分析

赵慧霞1(), 郭彦丽2(), 郑渝泠1, 何棱1, 陈锐1, 王珊珊1, 曾长立1, 邹珺3,4, 沈金雄3,4, 傅廷栋3, 刘小云1,*(), 万何平1,*()   

  1. 1 江汉大学生命科学学院, 湖北武汉 430056
    2 天津农学院园艺园林学院, 天津 300384
    3 华中农业大学作物遗传改良全国重点实验室, 湖北武汉 430070
    4 湖北国科高新技术有限公司, 湖北武汉 430070
  • 收稿日期:2025-04-10 接受日期:2025-08-13 出版日期:2025-11-12 网络出版日期:2025-08-18
  • 通讯作者: *万何平, E-mail: wanheping@jhun.edu.cn; 刘小云, E-mail: liuxiaoyun@jhun.edu.cn
  • 作者简介:赵慧霞, E-mail: zhaohuixia@jhun.edu.cn;
    郭彦丽, E-mail: feixueguoyanli@163.com

    **同等贡献

  • 基金资助:
    国家重点研发计划项目(2022YFD1201700);作物遗传改良全国重点实验室开放基金项目(ZK202403);国家自然科学基金项目(U22A20469)

Differential gene expression analysis of response to alkaline salt stress in Brassica napus L.

ZHAO Hui-Xia1(), GUO Yan-Li2(), ZHENG Yu-Ling1, HE Ling1, CHEN Rui1, WANG Shan-Shan1, ZENG Chang-Li1, ZOU Jun3,4, SHEN Jin-Xiong3,4, FU Ting-Dong3, LIU Xiao-Yun1,*(), WAN He-Ping1,*()   

  1. 1 School of Life Sciences, Jianghan University, Wuhan 430056, Hubei, China
    2 College of Horticulture and Landscape Architecture, Tianjin Agricultural University, Tianjin 300384, China
    3 National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, Hubei, China
    4 Hubei Guoke High-tech Co., Ltd., Wuhan 430070, Hubei, China
  • Received:2025-04-10 Accepted:2025-08-13 Published:2025-11-12 Published online:2025-08-18
  • Contact: *E-mail: wanheping@jhun.edu.cn; E-mail: liuxiaoyun@jhun.edu.cn
  • About author:

    **Contributed equally to this work

  • Supported by:
    National Key Research and Development Program of China(2022YFD1201700);National Key Laboratory of Crop Genetic Improvement Open Fund(ZK202403);National Natural Science Foundation of China(U22A20469)

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摘要：

为了研究碱胁迫条件下不同油菜材料的差异表达基因，为耐碱油菜品种选育和耐碱胁迫的分子机制探究提供参考，本研究选取耐碱油菜华油杂62 (H62)和不耐碱油菜中双11改良系(ZS11)为试验材料，设置对照和0.10% Na2CO3胁迫处理，利用RNA-Seq技术对萌发期的地上部和地下部的基因表达进行分析，通过生物信息学方法对差异基因的生物学功能和代谢途径进行研究，筛选可能参与碱胁迫调控的基因，了解油菜萌发期响应碱胁迫的分子机制。结果表明，与对照相比，0.10% Na2CO3胁迫下H62和ZS11地上部和地下部生长均显著受到抑制。转录组分析显示，在0.10% Na2CO3胁迫处理下，H62和ZS11地下部分别筛选出1860个和6358个上调表达基因，以及952个和6747个下调表达基因；H62和ZS11地上部分别筛选出3776个和5385个上调表达基因，以及1336个和3051个下调表达基因。ZS11的地上部与地下部差异表达基因(deferentially expressed genes, DEGs)数量均显著多于H62，尤其是地下部差异更大。GO和KEGG富集分析显示，2个品种中的DEGs显著富集于不同的GO功能与KEGG通路，表明其响应机制存在差异。H62主要通过谷胱甘肽代谢、醛固酮代谢、丙酮酸盐代谢、三羧酸循环和类黄酮合成等通路响应碱胁迫。ZS11碱胁迫后，离子通路、钾离子跨膜转运、ABC转运因子、DNA复制和蛋白酶体等通路发生显著变化。本研究通过建立耐碱/敏感油菜品种(系)的特异性转录调控图谱，揭示油菜萌发期耐碱胁迫的核心代谢通路及品种(系)特异性调控机制，为耐碱种质创制提供了关键候选基因和理论依据。

关键词: 油菜, 碱胁迫, 转录组分析, 差异表达基因, 谷胱甘肽代谢

Abstract:

To investigate differentially expressed genes (DEGs) involved in the alkali stress response and to provide insights for breeding alkali-tolerant rapeseed varieties, we selected the alkali-tolerant cultivar Huayouza 62 (H62) and the alkali-sensitive improved line Zhongshuang 11 (ZS11). Both were subjected to treatment with 0.10% Na2CO3. RNA-Seq technology was employed to analyze gene expression in shoots and roots during the germination stage. Bioinformatics tools were used to explore the biological functions and metabolic pathways of DEGs, aiming to identify genes potentially involved in alkali stress regulation and to elucidate the underlying molecular mechanisms during rapeseed germination. The results showed that 0.10% Na2CO3 stress significantly inhibited shoot and root growth in both H62 and ZS11 compared to the control. Transcriptome profiling revealed that, under this stress condition, 1860 and 6358 genes were up-regulated, while 952 and 6747 genes were down-regulated in the roots of H62 and ZS11, respectively. In shoots, 3776 and 5385 up-regulated genes, along with 1336 and 3051 down-regulated genes, were identified in H62 and ZS11, respectively. Notably, ZS11 exhibited a substantially larger number of DEGs than H62, especially in the roots. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses revealed that DEGs in the two varieties were significantly enriched in distinct GO terms and KEGG pathways, reflecting divergent molecular responses. In H62, DEGs were mainly enriched in pathways such as glutathione metabolism, aldosterone metabolism, pyruvate metabolism, the tricarboxylic acid (TCA) cycle, and flavonoid biosynthesis. In contrast, ZS11 showed significant enrichment in pathways related to ion transport, potassium transmembrane transport, ABC transporters, DNA replication, and the proteasome. Overall, this study establishes a transcriptional regulatory landscape for alkali-tolerant and alkali-sensitive rapeseed lines, identifies key metabolic pathways and genotype-specific regulatory mechanisms during germination under alkali stress, and provides valuable candidate genes and a theoretical foundation for developing alkali-tolerant germplasm.

Key words: Brassica napus L., alkali stress, transcriptome analysis, differentially expressed genes, glutathione metabolism

表1

qRT-PCR引物及序列"

基因编号
Gene ID		 基因名称
Gene name		 正向引物
Forward primer (5′-3′)		 反向引物
Reverse primer (5′-3′)
BnaA06G0368200ZS ABCB15 CGTTTCAGCTGCGAAGGC AGTCCCCCACGATCACCT
BnaA08G0116700ZS POT9 ATGGTCGGCTGCATTGCT AGCACAACAGCAGTCCCA
BnaA09G0619300ZS GSTU25 AGAGCCCGATTCTCCTCGA CCTGGCCAAGTCTCGTCG
BnaA10G0048800ZS GRXS11 GACCCCGAATGCCGAGAG CCGCCAATGAAGACGGCT
BnaC04G0010000ZS ABCB4 CTGGACAAGCTGCAGCCT GGCCTCGCAGGGTAAGTG
BnaC07G0076200ZS GORK GTGTCTCCGGCGACAACA GAACGACTCGGCCAAGCT
BnaC07G0430800ZS POT9 ATGCATACGGGACTGCGG CGACAGCTCCACCACGAG
BnaC07G0445600ZS LDOX ACCCGAAATGCCCTCAGC GCTGCAGACCAGGAACCA
BnaC02G0037200ZS BnaActin7 CTATCCTCCGTCTCGATCTCGC CTTAGCCGTCTCCAGCTCTTGC

图1

油菜萌发期碱处理浓度筛选与表型变化 A: 0.07%、0.10%和0.13% Na2CO3处理8 d后H62和ZS11的表型；B: 0.07%、0.10%和0.13% Na2CO3处理8 d后H62和ZS11的下胚轴长度；C: 0.07%、0.10%和0.13% Na2CO3处理8 d后H62和ZS11的主根长度。CK: 清水对照; H62: 华油杂62; ZS11: 中双11。**：P < 0.01。"

表2

转录组测序相关数据"

样品
Sample		 测序总读数
Total reads		 质控后读数
Clean reads		 质量值≥20碱基百分比
Q20 (%)		 质量值≥30碱基百分比
Q30 (%)		 GC含量
GC content (%)		 总对比率
Total mapped (%)
HCS1 41,783,020 41,783,020 96.89 92.23 46.02 92.69
HCS2 41,752,366 41,752,366 96.95 92.35 46.05 92.80
HCS3 41,752,258 41,752,258 96.79 91.99 46.63 93.20
HSR1 41,758,916 41,758,916 97.19 92.95 44.72 87.69
HSR2 42,266,306 42,266,306 96.91 91.67 40.32 47.15
HSR3 41,739,264 41,739,264 96.82 92.06 45.74 90.05
HTS1 41,785,450 41,785,450 96.68 91.69 47.48 92.56
HTS2 41,823,946 41,823,946 96.86 92.15 46.92 92.46
HTS3 41,795,376 41,795,376 96.74 91.81 46.30 92.45
HTR1 41,812,198 41,812,198 96.88 92.23 46.36 89.14
HTR2 41,810,942 41,810,942 96.80 92.00 45.90 85.03
HTR3 41,818,612 41,818,612 96.78 91.98 46.80 87.04
ZCS1 41,829,120 41,829,120 96.72 91.77 46.85 97.36
ZCS2 41,790,096 41,790,096 96.96 92.35 46.41 97.40
ZCS3 41,799,510 41,799,510 96.90 92.22 45.92 97.37
ZCR1 41,813,108 41,813,108 96.96 92.34 45.00 96.15
ZCR2 41,793,882 41,793,882 96.97 92.38 45.24 96.33
ZCR3 41,827,322 41,827,322 97.05 92.63 44.40 95.05
ZTS1 41,801,204 41,801,204 97.07 92.67 46.72 97.28
ZTS2 41,817,078 41,817,078 96.92 92.31 46.68 97.47
ZTS3 41,817,562 41,817,562 96.86 92.12 46.78 97.38
ZTR1 41,817,100 41,817,100 96.13 91.17 46.35 94.38
ZTR2 42,466,696 42,466,696 96.06 90.98 46.32 94.38
ZTR3 41,760,878 41,760,878 96.92 92.32 46.50 96.53

图2

差异表达基因数目统计和韦恩分析 A: 4个比较组差异表达基因的数量；B: 4个比较组差异表达基因的韦恩分析。缩写同表2。"

图3

差异表达基因显著富集的前20个GO条目 缩写同图1。"

附表1

差异基因KEGG富集"

类别
Category		 通路
Pathway		 基因比例
Gene ratio		 校正后P值
Adjusted P-value
H62地下部上调差异基因
H62 root up-regulated DEGs		 谷胱甘肽代谢Glutathione metabolism 44/458 5.13E-16
半胱氨酸与甲硫氨酸代谢Cysteine and methionine metabolism 44/458 3.87E-13
戊糖与葡萄糖醛酸相互转化Pentose and glucuronate interconversions 34/458 3.15E-08
光合生物的固碳Carbon fixation in photosynthetic organisms 27/458 4.63E-08
氮代谢Nitrogen metabolism 17/458 4.87E-06
2-氧代羧酸代谢2-Oxocarboxylic acid metabolism 23/458 1.63E-05
乙醛酸和二羧酸代谢Glyoxylate and dicarboxylate metabolism 23/458 1.67E-05
芥子油苷生物合成Glucosinolate biosynthesis 12/458 1.75E-05
丙氨酸、天门冬氨酸和谷氨酸代谢
Alanine, aspartate and glutamate metabolism		 19/458 3.49E-05
抗坏血酸和醛固酮代谢Ascorbate and aldarate metabolism 17/458 6.39E-05
氨基糖和核苷酸糖代谢Amino sugar and nucleotide sugar metabolism 28/458 1.81E-04
维生素B6代谢Vitamin B6 metabolism 7/458 1.31E-03
精氨酸生物合成Arginine biosynthesis 12/458 0.001945
丙酮酸盐合成Pyruvate metabolism 19/458 0.003671
三羧酸循环TCA cycle 15/458 0.003715
光合作用Photosynthesis 12/458 0.024255
硫代谢Sulfur metabolism 10/458 0.028677
牛磺酸和次牛磺酸代谢Taurine and hypotaurine metabolism 5/458 0.038469
光合作用的天线蛋白Photosynthesis-antenna proteins 6/458 0.042067
ZS11地下部上调差异基因
ZS11 root up-regulated DEGs		 蛋白酶体Proteasome 74/1946 9.90E-16
戊糖与葡萄糖醛酸相互转化Pentose and glucuronate interconversions 76/1946 5.91E-05
氨基糖和核苷酸糖代谢Amino sugar and nucleotide sugar 82/1946 1.90E-04
精氨酸生物合成 Arginine biosynthesis 30/1946 6.87E-03
光合生物的固碳Carbon fixation in photosynthetic organisms 49/1946 6.87E-03
DNA复制DNA replication 30/1946 0.019511
H62地下部下调差异基因
H62 root down-regulated DEGs		 植物昼夜节律Circadian rhythm-plant 10/187 1.74E-04
ZS11地下部下调差异基因
ZS11 root down-regulated DEGs		 角质、木栓质和蜡质合成Cutin, suberin and wax biosynthesis 31/1232 4.30E-08
氮代谢Nitrogen metabolism 30/1232 5.41E-06
半乳糖代谢Galactose metabolism 30/1232 1.38E-04
甘油酯代谢Glycerolipid metabolism 39/1232 5.56E-04
脂肪酸延伸Fatty acid elongation 26/1232 1.62E-03
ABC转运ABC transporters 16/1232 2.88E-03
精氨酸和脯氨酸代谢Arginine and proline metabolism 31/1232 3.48E-03
色氨酸代谢Tryptophan metabolism 27/1232 6.26E-03
ZS11地下部下调差异基因
ZS11 root down-regulated DEGs		 植物昼夜节律Circadian rhythm-plant 19/1232 0.045403
维生素B6代谢Vitamin B6 metabolism 9/1232 0.045694
丙氨酸、天冬氨酸和谷氨酸代谢
Alanine, aspartate and glutamate metabolism		 26/1232 0.045694
半胱氨酸和甲硫氨酸代谢Cysteine and methionine metabolism 45/1232 0.045694
H62地上部上调差异基因
H62 shoot up-regulated DEGs		 氨基糖和核苷酸糖代谢Amino sugar and nucleotide sugar metabolism 64/829 3.14E-13
谷胱甘肽代谢Glutathione metabolism 52/829 1.41E-11
脂肪酸延伸Fatty acid elongation 17/829 5.55E-04
半胱氨酸和甲硫氨酸代谢Cysteine and methionine metabolism 40/829 7.53E-04
黄酮类化合物生物合成Flavonoid biosynthesis 15/829 7.53E-04
抗坏血酸和醛固酮代谢Ascorbate and aldarate metabolism 20/829 8.28E-03
戊糖和葡萄糖醛酸相互转化Pentose and glucuronate interconversions 33/829 0.015446
氮代谢Nitrogen metabolism 16/829 0.019140
角质、木栓质和蜡质合成Cutin, suberine and wax biosynthesis 14/829 0.024177
半乳糖代谢Galactose metabolism 17/829 0.03368
H62地上部下调差异基因
H62 shoot down-regulated DEGs		 植物昼夜节律Circadian rhythm-plant 14/258 1.31E-06
光合生物的固碳Carbon fixation in photosynthetic organisms 13/258 7.11E-03
卟啉与叶绿素代谢Porphyrin and chlorophyll metabolism 9/258 0.010493
维生素B2代谢Riboflavin metabolism 5/258 0.016962
油菜素内酯合成Brassinosteroid biosynthesis 4/258 0.016962
氰基氨基酸代谢Cyanoamino acid metabolism 10/258 0.016962
氮代谢Nitrogen metabolism 8/258 0.016962
精氨酸和脯氨酸代谢Arginine and proline metabolism 10/258 0.017826
叶酸合成Folate biosynthesis 6/258 0.019124
ZS11地上部下调差异基因
ZS11 shoot down-regulated DEGs		 植物昼夜节律Circadian rhythm-plant 21/458 2.14E-08
精氨酸和脯氨酸代谢Arginine and proline metabolism 16/458 0.016201
醚脂代谢Ether lipid metabolism 9/458 0.016201
氮代谢Nitrogen metabolism 12/458 0.016201
类固醇生物合成Steroid biosynthesis 11/458 0.016201
卟啉与叶绿素代谢Porphyrin and chlorophyll metabolism 12/458 0.016351
甘油磷脂代谢Glycerolipid metabolism 17/458 0.023850
油菜素内酯合成Brassinosteroid biosynthesis 5/458 0.024615
糖胺聚糖降解Glycosaminoglycan degradation 4/458 0.027958
过氧物酶体Peroxisome 17/458 0.028641
半乳糖代谢Galactose metabolism 12/458 0.028641
倍半萜和三萜生物合成Sesquiterpenoid and triterpenoid biosynthesis 6/458 0.034004

图4

H62和ZS11特有和共有的差异基因集KEGG富集通路 A: H62地下部特有基因集; B: H62地上部特有基因集; C: ZS11地下部特有基因集; D: ZS11地上部特有基因集; E: H62和ZS11共有基因集。缩写同表2。"

图5

关键基因表达热图 A: 谷胱甘肽相关差异表达基因; B: 离子运输相关差异表达基因; C: 其他差异表达基因。缩写同表2。"

图6

部分差异表达基因qRT-PCR验证 缩写同表2。*、**、***、****分别表示在0.05、0.01、0.001和0.0001水平差异显著。ns表示差异不显著。"

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