基于转录组分析油菜素内酯对高温胁迫下酿酒葡萄花色苷合成及果实品质的调控机制

doi:10.3864/j.issn.0578-1752.2023.06.010

摘要/Abstract

摘要：

【目的】分析高温胁迫下参与油菜素内酯调控葡萄花色苷及果实品质合成的相关基因，探讨油菜素内酯调控果实花色苷及品质合成的机制。【方法】以酿酒葡萄‘赤霞珠’为试材，转色前一周利用红外辐射器模拟高温环境，并全树喷施0.6 mg∙L^-1的2,4-表油菜素内酯（2,4-Epibrassinolide，EBR），测定花色苷、总糖及相关品质指标，选择转色中期（花后70 d）的果实进行转录组测序，从分子水平阐述EBR对高温胁迫下花色苷合成的影响。【结果】从转色开始，各处理花色苷含量逐渐升高；成熟时，高温组（HT）花色苷总量显著低于对照组（CK），高温油菜素内酯组（HTE）花色苷含量高于HT组。总糖、还原糖、蔗糖变化规律与花色苷相似，HT组含量均在成熟期时低于CK组，成熟期各种糖含量为CK组>HTE组>HT组。分析3种处理下‘赤霞珠’果实基因水平的差异，通过GO和KEGG富集发现了14个与蔗糖和淀粉代谢途经相关的差异基因，其中HT和HTE处理显著上调了10个基因，显著下调了4个基因；苯丙氨酸代谢途径有11个差异基因，其中有7个参与花色苷合成的基因在HT处理中上调，有4个参与木质素合成的基因在HT处理下表达量显著上调，说明高温可能促进了木质素合成，从而降低了花色苷的积累量；内源激素信号转导途径的差异表达基因11个，其中ABA信号转导受体基因PP2C及激酶SnRK2的表达量在高温胁迫下显著提高，其可能与EBR共同参与调控高温胁迫下葡萄花色苷的合成。对部分差异基因的表达模式进行实时荧光定量（qRT-PCR）验证，证实了转录组数据的准确性。【结论】EBR通过改变糖代谢、花色苷合成及内源激素信号转导过程中相关基因的表达模式，缓解了高温胁迫对葡萄花色苷积累的抑制作用，提高了果实品质。

关键词: 酿酒葡萄‘赤霞珠’, 2,4-表油菜素内酯, 高温, 花色苷, 转录组

Abstract:

【Objective】 The aims of the study were to analyze the genes involved in the regulation of grape anthocyanin accumulation and fruit quality by 2,4-Epibrassinolide (EBR) under high-temperature stress, and to explore the molecular mechanism of EBR regulation anthocyanin accumulation in grapes under high-temperature stress. 【Method】 Cabernet Sauvignon grapes were treated with high-temperature stress using infrared emitter, and sprayed 0.6 mg∙L^-1 of EBR before the veraison. The content of total anthocyanins, total sugar, reducing sugar and sucrose were quantified using the ultraviolet visible spectrophotometer. The mechanism of EBR-mediated accumulation of anthocyanin under high-temperature stress was analyzed by transcriptome sequencing. 【Result】 Starting from veraison, the anthocyanin content increased gradually under various treatments. At maturity, the total anthocyanin content in the high temperature group (HT) was significantly lower than that in the control group (CK), and the anthocyanin content in the high temperature and EBR group (HTE) was higher than that in the HT group, but lower than CK group. Under HT treatment, the accumulation pattern of total sugar, reducing sugar and sucrose was similar to that of anthocyanins and lower than those of CK group at maturity stage. Compared with HT group, the contents of various sugars in HTE group were increased. The differences in transcriptome levels of Cabernet Sauvignon fruits under the three treatments were analyzed. Through GO and KEGG enrichment, 14 differential genes related to sucrose and starch metabolic pathways, among which 10 genes were significantly up-regulated and 4 genes were significantly down-regulated under HT and HTE treatments. The expressions of 11 genes were different in the phenylpropane metabolic pathway. Seven genes involved in anthocyanin synthesis were up-regulated under the HT treatment, and 4 genes involved in lignin synthesis were significantly up-regulated under the HT treatment, indicating that high temperature might promote lignin synthesis and reduce the accumulation of anthocyanins. In the endogenous hormone signaling pathway, the expression of the ABA signaling receptor genes PP2C and SnRK2 was significantly increased under high-temperature stress, and might be involved in regulating the synthesis of grape anthocyanin under high-temperature stress together with EBR. The expression patterns of some differential genes were verified by qRT-PCR, which confirmed the accuracy of transcriptome data. 【Conclusion】 EBR alleviated the inhibitory effect of high temperature stress on grapevine anthocyanin accumulation, probably due to the fact that EBR reduced the expression of lignin-related genes and changed the expression pattern of grape endogenous hormone signal transduction genes.

Key words: Vitis vinifera L. cv Cabernet Sauvignon, 2,4-Epibrassinolide, high-temperature, anthocyanin, RNA-seq

汪月宁, 代红军, 贺琰, 魏强, 郭学良, 刘妍, 殷梦婷, 王振平. 基于转录组分析油菜素内酯对高温胁迫下酿酒葡萄花色苷合成及果实品质的调控机制[J]. 中国农业科学, 2023, 56(6): 1139-1153.

WANG YueNing, DAI HongJun, HE Yan, WEI Qiang, GUO XueLiang, LIU Yan, YIN MengTing, WANG ZhenPing. Regulation Mechanism of Brassinolide on Anthocyanins Synthesis and Fruit Quality in Wine Grapes Under High Temperature Stress Based on Transcriptome Analysis[J]. Scientia Agricultura Sinica, 2023, 56(6): 1139-1153.

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基因名称 Gene name	正向引物 Forward primer (5′-3′)	反向引物 Reverse Primer (5′-3′)	NCBI登录号 Accession number
VvActin	CTTGCATCCCTCAGCACCTT	TCCTGTGGACAATGGATGGA	NB000705
VvF3′5′H	ACTAAGCCACAGGAAACTAA	AAACCGCTCAGACCAAAACC	LOC100243414
VvSS	CCAGCCAACGTCATTAGCCT	CCATGACTTGTGGCCTTCCT	LOC100252799
VvLDOX	GCCTAAGACACCAAGCGACTACG	CAACCCAAGCGATAGCACCGATAG	LOC100233142
VvLAR	TGCTTTTGTGATTTTGTTAGAGG	CCCTTCCCCGATTGAGAGTA	LOC100232982
VvINV	CCAGAAAACTTGTAGAAGCACC	GTTGACGCATTCCTTAAGGATC	LOC100232951
VvBZR1	GCACGCCATCTTGCTCCAT	AAGCTCCAGATCATCCAAACCTA	LOC100262472
VvF3′H	GGTGATGTTAGGCAGGAGAGTGTTC	GCTCCACCACCATCTCTTTGAACTC	LOC100232896
VvPP2C	CCACGCTTCCCTCAAACCCTCCCA	CATCCACCACTCCATCATCCCCGC	LOC100242943
VvSnRK2	CAATGCATGCTTTGAAAGTGTG	GCCAAACTGCAGTCTGATTTTA	LOC100260262

时间 Time	处理 Treatment	花色苷 Anthocyanins (mg∙L^-1)	总糖 TotalSugar (mg∙g^-1)	还原糖 Reducing sugar (mg∙g^-1)	蔗糖 Sucrose (mg∙g^-1)
7.20	CK	0.2±0.51a	14.61±1.92a	8.90±0.02a	7.12±0.15a
	HT	0.2±0.14a	14.56±2.02a	9.13±0.18a	7.35±0.26a
	HTE	0.2±0.28a	14.22±1.80a	9.14±0.74a	7.12±.028a
8.10	CK	12.91±0.86c	143.72±5.14a	20.71±1.61a	15.64±0.65b
	HT	14.30±0.79ab	124.72±3.52b	29.88±1.14a	16.56±0.48ab
	HTE	15.09±0.24a	137.35±6.55a	36.30±3.02a	16.92±0.54a
8.30	CK	35.24±2.11ab	199.21±4.55a	127.68±2.36a	23.85±5.48a
	HT	32.61±2.59c	174.96±0.52b	118.02±2.18b	19.92±2.61c
	HTE	36.04±2.98a	199.30±6.63a	124.29±9.47ab	22.34±0.69ab
9.20	CK	49.10±3.07a	225.87±4.19a	128.04±11.85a	26.67±6.93bc
	HT	39.33±2.75c	216.42±4.57b	108.17±15.81c	27.07±6.21b
	HTE	41.19±2.64b	222.41±5.16a	121.40±10.70b	29.43±5.35a

样品名称 Sample	原始序列数据 Raw reads (M)	原始序列长度 Raw bases (G)	过滤后数据 Clean reads (M)	过滤后序列长度 Clean bases (G)	比对率 Valid bases (%)	Q30 (%)	GC (%)
CK1	50.55	7.58	49.83	7.26	95.71	91.50	47.66
CK2	47.78	7.17	47.06	6.86	95.75	92.75	47.52
CK3	47.30	7.10	46.61	6.79	95.68	91.75	47.55
HT1	48.07	7.21	47.37	6.88	95.37	89.64	47.94
HT2	49.42	7.41	48.66	7.08	95.49	92.00	47.56
HT3	49.86	7.48	49.12	7.16	95.69	91.16	47.17
HTE1	48.84	7.33	48.09	7.01	95.71	92.44	47.45
HTE2	51.05	7.66	50.31	7.31	95.52	91.53	47.74
HTE3	50.46	7.57	49.75	7.25	95.78	92.67	47.37

处理 Treatment	差异转录因子个数 Transcription factor number	上调 Up	下调 Down	转录因子家族 Transcription factor family
HT vs CK	95	74	21	AP2、bHLH、bZIP、C2H2、Dof、ERF、HD-ZIP、LBD、MIKC-MADS、MYB、NAC、WRKY
HTE vs CK	57	44	13	bHLH、bZIP、Dof、ERF、HD-ZIP、LBD、MYB、NAC、WRKY
HT vs HTE	6	1	5	MYB