SO2引起巨峰葡萄采后落粒的共表达网络和转录调控分析

doi:10.3864/j.issn.0578-1752.210844

摘要/Abstract

摘要： 【目的】二氧化硫(SO₂)处理可有效防治灰霉病和采后腐烂，但会导致葡萄浆果脱落，研究SO₂引起葡萄浆果脱落的分子机制，明确关键基因和转录调控机制。【方法】使用SO₂处理葡萄后，分别在2、4和6 d采样并统计CK对照组和SO₂处理组的葡萄浆果脱落率，通过高通量测序技术对CK对照组和SO₂处理组‘巨峰’葡萄采后2、4、6 d的样品测序，以葡萄基因组作为参考基因组进行序列比对，利用TPM算法计算基因表达量，使用基因集富集分析(GSEA)、基因共表达网络(GCN)以及转录调控网络预测方法对转录组数据进行系统的分析，并利用实时荧光定量PCR(qRT-PCR)技术对其表达量进行验证。【结果】SO₂处理能够显著诱导葡萄浆果脱落，在2 d时SO₂处理的葡萄浆果脱落率为9.88%，在4 d时达到19.24%且均显著高于对照组，在6 d时脱落率达到38.25%，而对照组脱落率仅有11.85%。通过GSEA分析，发现在CK组富集的GO生物过程主要跟氧化应激反应，细胞壁代谢以及苯丙氨酸代谢通路相关，其中4 d时期CK组富集到植物细胞壁组织、果胶代谢、细胞壁修饰、聚半乳糖醛酸等。在SO₂组富集的GO生物过程主要跟能量代谢通路相关，其中2 d时在SO₂组富集到光合作用、四吡咯代谢、前体代谢物和能量的产生、葡萄糖代谢等。CK组富集的KEGG代谢通路主要有戊糖和葡萄糖醛酸的相互转换、半乳糖代谢、植物激素信号转导、柠檬酸循环（TCA循环）等，SO₂组有光合作用、柠檬酸循环（TCA循环）、糖酵解等。GCN将GSEA分析中的领头基因分为12个水平，水平4-水平9均富集到了能量代谢，糖代谢相关通路，其中水平4富集到了激素反应、氧化应激反应。对GCN关键level中的基因启动子序列进行转录调控预测分析，结果显示95个转录因子(TFs)与269个靶基因存在987对调控关系。WRKY14、WOX8、KUA1在SO₂处理下持续下调。MYB60、MYB73、ANL2、ERF2、DOF3.6、GATA25、WRKY57、KAN2、ATHB6在SO₂处理后持续上调。此外MYB15、WRKY11、WRKY33、WRKY40、WRK75先上调后下调。ERF2、MYB60和WRKY40的转录调控网络发现调控的靶基因涉及到细胞壁代谢、糖代谢等相关途径。qRT-PCR结果显示PME36和ERF2上调表达趋势相似，GAUT7、MYB60、UGE3上调表达趋势相似，此外WRKY40在SO₂处理的2和4 d被诱导上调，PPME1、COMT1表达持续下调，SO₂ 4 d时LAC15被显著诱导上调。【结论】SO₂诱导营养物质代谢、能量代谢、细胞壁代谢途径相关基因的表达，该过程受到多种转录因子调控，最终导致葡萄浆果脱落。

关键词: 葡萄, 脱落, GCN, 转录因子, 调控

Abstract: 【Objective】Sulfur dioxide (SO₂) treatment can effectively prevent Botrytis cinerea and postharvest decay, but it can lead to grape berry abscission. The purpose of this study was to clarify the key genes and transcriptional regulation mechanism. 【Method】After grapes were treated with SO₂, the grape berry abscission rates of CK control group and SO₂ treatment group were collected at 2, 4 and 6 days, respectively. Samples of ‘Kyoho’ grapes in CK control and SO₂-treated were sequenced by high-throughput sequencing technique. The grape genome was used as the reference genome for sequence alignment, and the gene expression was calculated by TPM algorithm. The transcriptome data were systematically analyzed by gene set enrichment analysis (GSEA), gene co-expression network (GCN) and transcription regulation network prediction, and the expression was verified using quantitative real-time fluorescence PCR (qRT-PCR). 【Result】SO₂ treatment could significantly induced grape berry abscission, which was significantly higher than that of the control (9.88%) at 2 d. The abscission rate reached 38.25% at 6 d compared to 11.85% in the control. Transcriptome analysis based on the annotated information of GO and KEGG databases and analyzed by GSEA software, it was found that SO₂-induced grape berry abscission involves plant hormone signal transduction, cell wall metabolism, glucose metabolism, phenylalanine metabolism, energy metabolism and other pathways. GCN divided the leading genes in GSEA analysis into 12 levels. Level 4 ~ 9 were enriched in energy metabolism and glucose metabolism-related pathways, level 4 was enriched in hormone response and oxidative stress response. The transcriptional regulation prediction analysis of transcriptional regulation of GCN key level gene promoter sequences showed that there were 987 pairs of regulatory relationships among 95 transcription factors (TFs). WRKY14, WOX8, KUA1 continued to be downregulated under SO₂ treatment. MYB60, MYB73, ANL2, ERF2, DOF3.6, GATA25, WRKY57, KAN2, ATHB6 continued to be upregulated under SO₂ treatment. In addition, MYB15, WRKY11, WRKY33, WRKY40, WRK75 were first adjusted upwards and then downwards. Mapping the transcriptional regulatory networks of ERF2, MYB60 and WRKY40 revealed that the regulated target genes were involved in cell wall metabolism, sugar metabolism and other related pathways. The qRT-PCR results showed that PME36 and ERF2 had similar up-regulated expression trends, GAUT7, MYB60 and UGE3 had similar up-regulated expression trends, in addition WRKY40 was induced to be up-regulated at 2 and 4 d of SO₂ treatment, PPME1 and COMT1 expression was consistently down-regulated, and LAC15 was significantly induced to be up-regulated at 4 d of SO₂.【Conclusion】SO₂-induced the expression of genes related to nutrient metabolism, energy metabolism and cell wall metabolism pathway, which was regulated by a variety of transcription factors, and eventually led to the grape berry abscission.

Key words: grape, abscission, GCN, Transcription factor, regulation

杨盛迪, 孟祥轩, 郭大龙, 裴茂松, 刘海楠, 韦同路, 余义和. SO₂引起巨峰葡萄采后落粒的共表达网络和转录调控分析[J]. 中国农业科学, 0, (): 1-.

YANG ShengDi, MENG XiangXuan, GUO DaLong, LIU HaiNan, PEI MaoSong, WEI TongLu, YU YiHe. Co-expression Network and Transcriptional Regulation Analysis of SO₂-induced Postharvest Abscission of ‘Kyoho’ Grape[J]. Scientia Agricultura Sinica, 0, (): 1-.

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SO₂引起巨峰葡萄采后落粒的共表达网络和转录调控分析

Co-expression Network and Transcriptional Regulation Analysis of SO₂-induced Postharvest Abscission of ‘Kyoho’ Grape

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