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.0578-1752.2022.11.011

摘要/Abstract

摘要：

【目的】二氧化硫（SO₂）处理可有效防治葡萄灰霉病和采后腐烂,但会导致浆果脱落,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‘巨峰’葡萄,分别在2、4和6 d进行采样并统计对照组（CK）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和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,2 d时,SO₂处理的葡萄浆果脱落率为9.88%,4 d时达到19.24%,且均显著高于对照组,6 d时脱落率达到38.25%,而对照组脱落率仅11.85%。通过GSEA分析,发现在CK组富集的GO生物过程主要与氧化应激反应、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、聚半乳糖醛酸等通路。在SO₂组富集的GO生物过程主要与能量代谢通路相关,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、葡萄糖代谢等通路。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—9均富集到了能量代谢、糖代谢相关通路,其中水平4富集到激素反应、氧化应激反应。对GCN关键水平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处理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 berry abscission, the purpose of this study was to explore the molecular mechanism of grape berry abscission induced by SO₂. 【Method】 Kyoho grapes were treated with SO₂, samples were collected at 2, 4 and 6 d, respectively, and the grape berry abscission rates of the control group (CK) and SO₂ treatment group. The samples of ‘Kyoho’ grapes under CK control and SO₂-treated were sequenced at 2, 4 and 6 d after harvest 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 by using quantitative real-time fluorescence PCR (qRT-PCR). 【Result】 SO₂ treatment could significantly induce grape berry abscission, the grape berry abscission treated with SO₂ was 9.88% at 2 d and 19.24% at 4 d, which were significantly higher than those in the control group, the abscission rate reached 38.25% at 6 d, while the abscission rate of the control group was only 11.85%. GSEA analysis showed that the GO biological process enriched in CK group was mainly related to oxidative stress response, cell wall metabolism and phenylalanine metabolic pathway, and CK group was enriched in plant cell wall tissue, pectin metabolism, cell wall modification, polygalacturonic acid and other pathways. The GO biological process enriched in SO₂ group is mainly related to energy metabolism pathway, and it is enriched to photosynthesis, tetrapyrrole metabolism, precursor metabolite and energy production, glucose metabolism and other pathways in SO₂ group at 2 d. The KEGG metabolic pathways enriched in CK group mainly include the mutual conversion of pentose and glucuronic acid, galactose metabolism, plant hormone signal transduction, citric acid cycle (TCA cycle), etc. SO₂ group includes photosynthesis, citric acid cycle (TCA cycle), glycolysis, etc. GCN divided the leading genes in GSEA analysis into 12 levels: level 4-9 were enriched in energy metabolism and glucose metabolism-related pathways, while level 4 was enriched in hormone response and oxidative stress response. The transcriptional regulation prediction analysis of GCN key level gene promoter sequences showed that there were 987 pairs of regulatory relationships among 95 transcription factors (TFs). WRKY14, WOX8, KUA1 were continuously downregulated under SO₂ treatment, wihle MYB60, MYB73, ANL2, ERF2, DOF3.6, GATA25, WRKY57, KAN2, ATHB6 were continuously upregulated under SO₂ treatment. In addition, MYB15, WRKY11, WRKY33, WRKY40, WRK75 were first adjusted upwards and then downwards. 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 the up-regulated expression trend of PME36 and ERF2 was similar, and 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 up-regulated at 4 d of SO₂ treatment. 【Conclusion】 SO₂induced 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 leads 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, 2022, 55(11): 2214-2226.

YANG ShengDi,MENG XiangXuan,GUO DaLong,PEI MaoSong,LIU HaiNan,WEI TongLu,YU YiHe. Co-Expression Network and Transcriptional Regulation Analysis of Sulfur Dioxide-Induced Postharvest Abscission of Kyoho Grape[J]. Scientia Agricultura Sinica, 2022, 55(11): 2214-2226.

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基因名称 Gene name	上游引物Primer sequence (5′-3′)	下游引物 Primer sequence (5′-3′)
Vitvi18g03065:LAC15	CCGAGCCACTGTTCATGGAG	AGCACTTCATCTCGAACCGC
Vitvi11g00034:PPME1	GTGATCACAGCTCAGGCAAG	TGAGAGTGCCCATGTAGGTG
Vitvi09g01122:WRKY40	CTGCCCTGTCAAGAAGAAGG	TGAGCTGAGTGAGGATGCAC
Vitvi16g02061:COMT1	CGAAGGGCCGGCCAC	CAGGGGAGGGGATGTCTCTT
Vitvi16g00349:ERF2	CAGTCTGATGCCGTTCTTGA	CCTAGGCTCCTCCTTCACCT
Vitvi15g00703:PME36	GAAGGAAGTGCAACCGACAT	TATGGACCATGCGGGTTAGT
Vitvi01g01838:UPTG2	CTGGCAAGAAGAGCTGATCC	AATCTCCAGCAGATGGGTTG
Vitvi08g00069:MYB60	CAGGCCTAAGTTGGAGCAAG	GGAGGGTTGTGCTTCTTCTG
Vitvi15g00687:UGE3	GTGGGCTTCTTCGAGTTCAG	CCAGTAAGCGAAAGGCCATA
Vitvi08g00009:GAUT7	CATTGCCTCAGGAGTTTCGT	CCCAACCTCACCCTACAGAA
ubiqutin1	GTGGTATTATTGAGCCATCCTT	AACCTCCAATCCAGTCATCTAC

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 Sulfur Dioxide-Induced Postharvest Abscission of Kyoho Grape

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