Transcriptome Analysis of Hormone Signal Transduction and Glutathione Metabolic Pathway in Rice Seeds at Germination Stage

doi:10.16819/j.1001-7216.2021.200915

Abstract

Abstract:

【Objective】By using transcriptome sequencing technology, we explored the regulation mechanism of hormone signal transduction and redox balance inside cells during rice germination, as a way to increase the understanding of and construct the complex regulatory network of germination. At the same time, mining the genes that regulate seed germination could lay a theoretical basis for direct-seeded rice breeding.【Method】Dynamic transcriptome sequencing analysis was performed using seeds at 0, 24, and 48 hours after imbibition as materials. Differentially expressed genes (DEGs) were screened with the threshold of Fold Change≥2 and False Discovery Rate≤ 0.05. Gene Ontology and KEGG Pathway databases were used to analyze and annotate the DEGs at different stages of germination and real-time quantitative PCR was conducted to verify the sequencing results. Finally, the protein interaction network of DEGs was analyzed using the String protein interaction database under the threshold of combined_score≥0.9.【Result】8719 DEGs were identified in the early stage of seed germination, but only 3480 DEGs in the late stage of germination. The results of GO and KEGG enrichment analysis showed that the DEGs related to hormone signal transduction were mainly induced in the early stage, especially the GH3 (Gretchen Hagen 3) family genes in the auxin signal transduction pathway were all significantly induced after imbibition. While the DEGs involved in the glutathione metabolism pathway were more active during the late germination period, and the glutathione-S-transferase genes were the most enriched in this pathway. In addition, two isocitrate dehydrogenase (ICDH) genes were significantly induced throughout the germination process. According to protein interaction prediction, the two ICDH genes may interact with the GH3 family genes.【Conclusion】The GH3 family genes in the AUX signal transduction pathway may play an important role in attenuating the AUX signal and reducing the AUX activity in the early stage of seed germination. Their high expression levels reduce the regulatory effect of auxin on seed dormancy and promote seed germination. In the glutathione metabolism pathway, GST genes may play a major role in resistance to cell oxidative stress in the late stage of seed germination. In addition, the interaction between GH3 and ICDH family genes during the germination process may have important significance in realizing the tandem effect of hormone transduction pathway and glutathione metabolism pathway and co-regulating seed germination.

Key words: rice, germination, transcriptome, hormone, glutathione

摘要：

【目的】利用转录组测序技术,探究水稻萌发过程中激素信号转导和细胞内部氧化还原平衡的调控机理,以期增加对萌发过程中复杂调控机制的理解,促进萌发期基因组转录调控网络的构建,并挖掘调控种子萌发的相关基因,为水稻直播稻新品种选育提供理论参考。【方法】利用萌发0、24和48 h的种子进行动态转录组测序分析,以差异倍数≥2、错误发现率≤0.05为阈值筛选差异基因,并利用Gene Ontology(GO)和KEGG Pathway数据库对萌发不同阶段的差异基因进行分析注释;同时利用实时荧光定量PCR对测序结果进行验证;最后运用String蛋白互作数据库以combined_score≥0.9为阈值分析差异基因的蛋白互作网络。【结果】在种子萌发前期鉴定到8719个差异基因,而在萌发后期仅鉴定到3480个。GO和KEGG富集结果均显示与激素信号转导相关的基因主要在萌发前期被诱导,特别是生长素信号转导途径中的GH3家族基因在萌发前期均受到显著诱导;而谷胱甘肽代谢途径中的基因在萌发后期转录更为活跃,其中谷胱甘肽-S-转移酶基因富集最多。此外,两个异柠檬酸脱氢酶基因在萌发过程中被显著诱导,经蛋白互作预测发现两个异柠檬酸脱氢酶基因与GH3家族基因可能存在相互作用。【结论】在种子萌发前期,生长素信号转导途径中的GH3家族基因可能在减弱生长素信号以及降低生长素活性方面发挥着重要作用,其高表达能降低生长素对种子的休眠作用,促进萌发启动;在种子萌发后期,谷胱甘肽代谢途径中的谷胱甘肽-S-转移酶基因可能在细胞抵抗氧化胁迫中发挥主要作用;此外,在整个萌发过程中,GH3和异柠檬酸脱氢酶家族基因的相互作用可能在实现激素转导途径和谷胱甘肽代谢途径的交互串联作用、共同调控种子萌发方面具有重要意义。

关键词: 水稻, 萌发, 转录组, 激素, 谷胱甘肽

Huan CUI, Qiaoli GAO, Lixin LUO, Jing YANG, Chun CHEN, Tao GUO, Yongzhu LIU, Yongxiang HUANG, Hui WANG, Zhiqiang CHEN, Wuming XIAO. Transcriptome Analysis of Hormone Signal Transduction and Glutathione Metabolic Pathway in Rice Seeds at Germination Stage[J]. Chinese Journal OF Rice Science, 2021, 35(6): 554-564.

崔欢, 高巧丽, 罗立新, 杨靖, 陈淳, 郭涛, 刘永柱, 黄永相, 王慧, 陈志强, 肖武名. 水稻萌发期激素信号转导和谷胱甘肽代谢转录分析[J]. 中国水稻科学, 2021, 35(6): 554-564.

Figures/Tables 8

Table 1 Sequences of the primers used in qRT-PCR.

序号 No.	基因号 Gene ID	正向引物（5′-3′） Forward primer（5′-3′）	反向引物（3′-5′） Reverse primer（3′-5′）
1	Os01g0764800	TGATCACTCACTACACTACACG	ACACTGACACCGACTGTATAAG
2	Os07g0592600	CCTCTTCCTCTCGCTACTACTA	TCTCAACCCCGAACAAGAAAAA
3	Os05g0143800	ATACTTCGAGTTCATCCCGTTC	CCGACTTTGTACCGGTACAG
4	Os11g0528700	AACATGTGCTACTACGAGTTCA	GTACAACCCTGTGAAGGTGG
5	Os01g0370200	CGAGGCCATCTATCAAGAAGAT	CTTTTGTTACGAGGCACAAGAA
6	Os03g0283100	AAGATTGTCGCGATTGATCTTG	TGATTGTTGTGCTCAAGTGAAG
7	Os10g0528300	CAAGATCTTCGACGAGGAGAAG	CTCATCTTAGCGAACTCGACC
8	Os10g0529300	GACCTCCACAACAAGAGTGAG	AACTTGTCATTGATGTAGGCGG
9	Os03g0718100	GAATGCTAAGCCAAGAGGAG	AATCACAAGTGAGAACCACAG

Fig. 1. Differentially expressed gene analysis during different germination stages. A, MA map of DEG in early germination; B, MA map of DEG in late germination; C, Venn diagram of DEG in germination stage; D, Venn diagram of up-regulation and down-regulation DEG in germination stage. C0, 0 h after germination; C1, 24 h after germination; C2, 48 h after germination.

Fig. 2. GO enrichment plots of differentially expressed genes at early(A) and late(B) germination stages.

Fig. 3. KEGG enrichment plots of DEGs at different germination stages. C0, 0 h after germination; C1, 24 h after germination; C2, 48 h after germination.

Fig. 4. Heatmap of DEGs expression in hormone signal transduction pathways.

Fig. 5. Heatmap of DEGs expression in glutathione metabolic pathway.

Fig. 6. Predicted interaction networks between hormone signaling transduction pathways and glutathione metabolic pathways.

Fig. 7. Expression patterns of selected DEG were verified by qRT-PCR. C0, 0 h after germination; C1, 24 h after germination; C2, 48 h after germination. FPKM, Fragments per kilobase per million.

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