基于转录组分析ABA促进葡萄花青苷积累相关基因

doi:10.3864/j.issn.0578-1752.2022.01.012

摘要/Abstract

摘要：

【目的】分析参与调控ABA促进葡萄着色的相关基因,探讨ABA促进葡萄果实花青苷积累的分子机制。【方法】以‘红巴拉多’葡萄为试材,在转色前期（约花后6周）使用300 mg∙L^-1 ABA对果穗进行浸果处理,以清水处理为对照。观察表型,并利用超高液相色谱质谱联用仪（UPLC-MS）测定花青苷组分及含量,再利用转录组测序技术从分子水平对ABA促进花青苷积累的机制进行生物信息学分析。【结果】外源ABA处理3 d后,葡萄果实着色明显加深,花青苷种类和含量增多,其中芍药素3-O-葡萄糖苷、锦葵色素3-O-葡萄糖苷两种单体花青苷含量增加最为显著。分析ABA处理18 h和3 d后葡萄果实转录水平的差异,并通过KEGG富集分析发现了11个ABA信号通路基因以及52个与花青苷生物合成和转运相关的基因差异表达,它们均在外源ABA处理后表达上调。通过将差异基因与葡萄转录因子库进行比对,共发现297个转录因子差异表达。进一步分析差异转录因子表达模式,筛选与VvMYBA1表达模式相近的转录因子,发现15个MYB、bHLH、bZIP、NAC、Dof、HD-ZIP等家族的转录因子,其可能参与调控花青苷生物合成。启动子顺式作用元件分析表明,大部分筛选到的差异基因启动子中含有ABRE元件,说明这些差异表达基因的启动子可能为ABA诱导型启动子。对部分候选基因的表达模式进行实时荧光定量（qRT-PCR）分析,证实了RNA-seq的准确性。【结论】ABA促进葡萄花青苷积累涉及11个ABA信号转导、52个花青苷合成和转运相关基因,15个转录因子可能参与调控了这一生物过程。研究结果为揭示ABA促进葡萄花青苷积累的分子机制提供了一定基础。

关键词: 葡萄, 花青苷, ABA, 转录组, 启动子

Abstract:

【Objective】 The aim of this study was to analyze the genes involved in the regulation of ABA induced grape coloring, and to explore the molecular mechanism of ABA induced anthocyanin accumulation in grape. 【Method】 In present study, Benibalado was used as the experimental material. In the early stage of veraison, the grape clusters were treated with 300 mg·L^-1ABA, water treated as control. The grape phenotypes were observed and anthocyanins were determined by UPLC-MS. The mechanism of ABA promoting anthocyanin accumulation was analyzed by transcriptome sequencing. 【Result】 After 3 days of exogenous ABA treatment, the grape berries were obviously colored, and the variety and content of anthocyanins were also increased. Among them, Peonidin 3-O-glucoside and Malvidin 3-O-glucoside increased most significantly. By KEGG enrichment analysis, 11 DEGs related to ABA signaling and 52 DEGs that related to anthocyanin biosynthesis, and transportation were identified, all of which were up-regulated. After exogenous ABA treatment, the DEGs from RNA-seq were searched by using BLAST against the grape TF database, and 297 transcription factors were identified. Through the further analyzing of the expression patterns of identified TFs, 15 members of MYB, bHLH, bZIP, NAC, Dof, and HD-ZIP families were observed to regulate anthocyanin biosynthesis. The analyzing of cis-acting elements in promoters showed that ABREs were identified in most of the promoters. The accuracy of RNA-seq was validated by qRT-PCR analysis of some candidate genes. 【Conclusion】 Overall, ABA promoting anthocyanin accumulation in grape was a complex process, including 11 DEGs related to ABA signal transduction, 52 DEGs that related to anthocyanin biosynthesis, modification and transportation, 15 transcription factors. This study provided a basis for revealing the molecular mechanism of ABA promoting anthocyanin accumulation in grape fruits.

Key words: grape, anthocyanin, ABA, RNA-seq, promoter

徐献斌,耿晓月,李慧,孙丽娟,郑焕,陶建敏. 基于转录组分析ABA促进葡萄花青苷积累相关基因[J]. 中国农业科学, 2022, 55(1): 134-151.

XU XianBin,GENG XiaoYue,LI Hui,SUN LiJuan,ZHENG Huan,TAO JianMin. Transcriptome Analysis of Genes Involved in ABA-Induced Anthocyanin Accumulation in Grape[J]. Scientia Agricultura Sinica, 2022, 55(1): 134-151.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2022.01.012

https://www.chinaagrisci.com/CN/Y2022/V55/I1/134

图/表 13

表1

图1

表2

图2

图3

表3

图4

图5

图6

图7

图8

表4

差异基因2000 bp启动子序列中ABRE作用元件数量统计"

NCBI登录号 Accession number	FPKM值 FPKM Value				ABRE元件数量 The number of ABREs	基因注释 Gene annotation
NCBI登录号 Accession number	ABA18 h	CK18 h	ABA3 d	CK3 d	ABRE元件数量 The number of ABREs	基因注释 Gene annotation
LOC100233012	22.86	2.99	146.17	11.75	2	PAL
LOC100241575	27.27	3.94	146.49	10.51	1	PAL
LOC100241377	15.24	5.23	13.39	7.70	4	PAL
LOC100854997	1.09	0.24	1.48	0.87	6	PAL
LOC100245997	0.94	0.48	1.43	0.35	6	PAL
LOC100253493	72.53	6.17	23.01	2.61	3	C4H
LOC100267215	247.58	1.83	32.27	1.09	1	C4H
LOC100254698	32.99	12.71	23.72	13.25	4	4CL
LOC100245991	25.27	12.93	82.87	30.48	4	4CL
LOC100253166	1.81	0.76	0.54	0.39	3	CHS
LOC100263437	3.02	0.16	3.19	0.91	3	CHS
LOC100258106	843.21	485.95	3031.14	577.81	4	CHS
LOC100262321	3.25	1.53	3.78	1.58	3	CHS
LOC100264844	3.21	1.77	5.39	2.38	1	CHS
LOC100232843	187.92	57.83	509.38	113.44	0	CHS
LOC100263443	19.00	5.73	54.48	10.52	5	CHS
LOC100241164	2.14	0.39	1.66	1.34	0	CHS
LOC100233078	410.22	213.33	646.48	305.87	4	CHI
LOC100255217	255.72	183.82	637.94	284.91	4	CHI
LOC100232999	87.38	67.10	165.37	80.38	0	F3'H
LOC100261319	5.14	0.55	15.12	2.11	4	F3'5'H
LOC109122765	2.03	0.43	3.94	0.30	2	F3'5'H
LOC100243414	1.36	0.11	3.70	0.32	4	F3'5'H
LOC104877273	1.45	0.28	4.23	0.52	4	F3'5'H
LOC100241335	3.21	0.40	4.88	0.60	2	F3'5'H
LOC100233079	174.69	30.14	459.98	62.82	4	F3H
LOC100253950	526.93	291.78	592.54	357.45	5	F3H
LOC100233141	245.66	67.49	320.10	94.11	3	DFR
LOC100233142	493.12	246.34	1503.28	488.51	5	LDOX
LOC100233099	410.21	94.82	699.90	221.37	1	UFGT
LOC100852631	11.36	5.04	5.14	1.38	3	UFGT
LOC100264341	10.81	3.90	3.13	1.01	0	UFGT
LOC100257268	31.65	18.50	4.13	2.95	3	UFGT
LOC100255538	10.79	7.27	4.29	1.60	4	UFGT
LOC100265929	1.54	0.62	0.88	0.30	2	UFGT
NCBI登录号 Accession number	FPKM值 FPKM Value				ABRE元件数量 The number of ABREs	基因注释 Gene annotation
NCBI登录号 Accession number	ABA18 h	CK18 h	ABA3 d	CK3 d	ABRE元件数量 The number of ABREs	基因注释 Gene annotation
LOC100854172	103.23	23.28	67.84	21.74	1	OMT
LOC100233134	70.74	7.07	203.04	22.29	5	FAOMT
LOC100232862	105.44	36.52	55.05	25.23	2	OMT
LOC100232921	10.99	3.31	16.53	4.91	2	OMT
LOC100251744	6.90	3.62	4.87	3.39	0	OMT
LOC100243978	418.54	100.61	705.85	203.97	6	OMT
LOC100242506	38.68	4.88	72.67	6.66	0	GST
LOC100264838	8.74	1.18	3.02	0.60	4	GST
LOC100245065	19.94	8.57	17.88	14.00	4	GST
LOC100232976	190.44	68.50	1357.36	115.94	0	GST4
LOC100233043	231.50	154.62	241.01	199.76	0	GST
LOC100852746	3.00	1.06	1.65	1.09	1	GST
LOC104878920	5.29	1.65	5.94	1.69	1	3AT
LOC100252075	9.37	6.63	5.60	4.10	1	ABC
LOC100268149	75.98	92.38	114.89	97.89	11	VvAM1
LOC100250967	162.57	41.82	545.35	96.32	3	MATE
LOC100255800	16.82	2.83	25.14	9.63	5	MATE1
LOC100250616	38.07	11.73	37.19	17.00	2	MATE2
LOC100233098	31.24	2.48	39.68	9.14	13	MYBA1
LOC100232838	93.45	8.01	132.29	18.05	6	MYBA2
LOC100853472	166.39	8.79	333.69	23.14	6	MYBA3
LOC100233136	100.72	69.01	108.09	99.71	2	MYBC2-L1
LOC100254518	79.92	36.44	120.88	62.90	1	MYB4
LOC100251098	46.68	29.43	47.02	37.98	1	MYC1
LOC100260656	5.43	1.27	3.16	1.26	6	MYB
LOC100250940	6.10	1.08	2.67	1.14	2	MYB
LOC100260647	3.70	0.34	4.18	0.97	3	MYB
LOC104879728	34.16	7.04	18.04	4.46	8	NAC
LOC100243434	61.74	40.82	53.52	33.84	2	ABF1
LOC100232889	29.63	18.76	36.56	22.09	6	ABF2
LOC100258873	108.20	55.20	113.90	72.46	2	bZIP
LOC100265549	37.85	17.70	25.76	15.81	5	Dof
LOC100245524	51.19	6.03	32.12	5.14	6	HD-ZIP

表4

图9

参考文献 45

[1]	MEDOUNI-ADRAR S, BOULEKBACHE-MAKHLOUF L, CADOT Y, MEDOUNI-HAROUNE L, DAHMOUNE F, MAKOUKHE A, MADANI K. Optimization of the recovery of phenolic compounds from Algerian grape by products. Industrial Crops and Products, 2015, 77: 123-132. doi: 10.1016/j.indcrop.2015.08.039
[2]	PEPPI M C, FIDELIBUS M W, DOKOOZLIAN N K. Application timing and concentration of abscisic acid affect the quality of ‘Redglobe’ grapes. The Journal of Horticultural Science and Biotechnology, 2007, 82(2): 304-310. doi: 10.1080/14620316.2007.11512233
[3]	AMIRI M E, FALLAHI E, MIRJALILI M. Effects of abscisic acid or ethephon at veraison on the maturity and quality of ‘Beidaneh Ghermez’ grapes. The Journal of Horticultural Science and Biotechnology, 2009, 84(6): 660-664. doi: 10.1080/14620316.2009.11512582
[4]	VILLALOBOS-GONZÁLEZ L, PEÑA-NEIRA A, IBÁÑEZ F, PASTENES C. Long-term effects of abscisic acid (ABA) on the grape berry phenylpropanoid pathway: Gene expression and metabolite content. Plant Physiology and Biochemistry, 2016, 105: 213-223.
[5]	FORLANI S, MASIERO S, MIZZOTTI C. Fruit ripening: The role of hormones, cell wall modifications, and their relationship with pathogens. Journal of Experimental Botany, 2019, 70(11): 2993-3006. doi: 10.1093/jxb/erz112
[6]	CELIA C M, FIDELIBUS M W, CRISOSTO C H. Application of abscisic acid (ABA) at veraison advanced red color development and maintained postharvest quality of ‘Crimson Seedless’ grapes. Postharvest Biology and Technology, 2007, 46(3): 237-241. doi: 10.1016/j.postharvbio.2007.05.017
[7]	李芳菲, 王莎, 谷世超, 程大伟, 顾红, 李明, 陈锦永, 杨英军. 叶面喷施ABA和PDJ对‘巨峰’葡萄果实着色及品质的影响. 果树学报, 2020, 37(3): 362-370.
	LI F F, WANG S, GU S C, CHENG D W, GU H, LI M, CHEN J Y, YANG Y J. Effects of foliar application of ABA and PDJ on the coloration and quality of ‘Kyoho’ grape berry. Journal of Fruit Science, 2020, 37(3): 362-370. (in Chinese)
[8]	张培安, 王壮伟, 蔡斌华, 文习成, 田亮, 王晨, 贾海锋, 房经贵. ABA对‘巨峰’葡萄采后成熟关键基因表达的影响. 园艺学报, 2018, 45(6): 1067-1080.
	ZHANG P A, WANG Z W, CAI B H, WEN X C, TIAN L, WANG C, JIA H F, FANG J G. Effects of ABA on the expression of key genes in postharvest fruit of ‘Kyoho’ grapevine. Acta Horticulturae Sinica, 2018, 45(6): 1067-1080. (in Chinese)
[9]	马文瑶, 程大伟, 顾红, 黄海娜, 陈锦永, 杨英军. 脱落酸(ABA)促进果实着色研究进展. 果树学报, 2018, 35(8): 1016-1026.
	MA W Y, CHENG D W, GU H, HUANG H N, CHEN J Y, YANG Y J. Advances in ABA promoting fruit coloration. Journal of Fruit Science, 2018, 35(8): 1016-1026. (in Chinese)
[10]	KATAYAMA-IKEGAMI A, SAKAMOTO T, SHIBUYA K, KATAYAMA T, GAO-TAKAI M. Effects of abscisic acid treatment on berry coloration and expression of flavonoid biosynthesis genes in grape. American Journal of Plant Sciences, 2016, 7(9): 1325-1336. doi: 10.4236/ajps.2016.79127
[11]	KOYAMA R, ROBERTO S R, DE SOUZA R T, BORGES W F S, ANDERSON M, WATERHOUSE A L, CANTU D, FIDELIBUS M W, BLANCO-ULATE B. Exogenous abscisic acid promotes anthocyanin biosynthesis and increased expression of flavonoid synthesis genes in Vitis vinifera × Vitis labrusca table grapes in a subtropical region. Frontiers in Plant Science, 2018, 9: 323. doi: 10.3389/fpls.2018.00323
[12]	GAO Z, LI Q, LI J, CHEN Y J, LUO M, LI H, WANG J Y, WU Y S, DUAN S Y, WANG L, SONG S R, XU W P, ZHANG C X, WANG S P, MA C. Characterization of the ABA receptor VlPYL1 that regulates anthocyanin accumulation in grape berry skin. Frontiers in Plant Science, 2018, 9: 592. doi: 10.3389/fpls.2018.00592
[13]	JIA H R, ZHANG Z B, ZHANG S H, FU W H, SU L Y, FANG J G, JIA H F. Effect of the methylation level on the grape fruit development process. Journal of Agricultural and Food Chemistry, 2020, 68(7): 2099-2115. doi: 10.1021/acs.jafc.9b07740
[14]	ZHANG L, XU Y S, JIA Y, WANG J Y, YUAN Y, YU Y, TAO J M. Effect of floral cluster pruning on anthocyanin levels and anthocyanain-related gene expression in ‘Houman’ grape. Horticulture Research, 2016, 3: 16037. doi: 10.1038/hortres.2016.37
[15]	HU B, LAI B, WANG D, LI J Q, CHEN L H, QIN Y Q, WANG H C, QIN Y H, HU G B, ZHAO J T. Three LcABFs are involved in the regulation of chlorophyll degradation and anthocyanin biosynthesis during fruit ripening in Litchi chinensis. Plant and Cell Physiology, 2018, 60(2): 448-461. doi: 10.1093/pcp/pcy219
[16]	LIVAL K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR. Methods, 2002, 25(4): 402-408. doi: 10.1006/meth.2001.1262
[17]	CASTELLARIN S D, GASPERO G D, MARCONI R, NONIS A, PETERLUNGER E, PAILLARD S, ADAM-BLNODON A F, TESTOLIN R. Colour variation in red grapevines (Vitis vinifera L.): Genomic organisation, expression of flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase genes and related metabolite profiling of red cyanidin-/blue delphinidin-based anthocyanins in berry skin. BMC Genomics, 2006, 7: 12. doi: 10.1186/1471-2164-7-12
[18]	CRUPI P, ALBA V, MASI G, CAPUTO A R, TARRICONE L. Effect of two exogenous plant growth regulators on the color and quality parameters of seedless table grape berries. Food Research International, 2019, 126: 108667. doi: 10.1016/j.foodres.2019.108667
[19]	GIRIBALDI M, GÉNY L, DELROT S, SCHUBERT A. Proteomic analysis of the effects of ABA treatments on ripening Vitis vinifera berries. Journal of Experimental Botany, 2010, 61(9): 2447-2458. doi: 10.1093/jxb/erq079
[20]	LIOTENBERG S, NORTH H, MARION-POLL A. Molecular biology and regulation of abscisic acid biosynthesis in plants. Plant Physiology and Biochemistry, 1999, 37(5): 341-350. doi: 10.1016/S0981-9428(99)80040-0
[21]	ZHANG M, LENG P, ZHANG G L, LI X X. Cloning and functional analysis of 9-Cis-epoxycarotenoid dioxygenase (NCED) genes encoding a key enzyme during abscisic acid biosynthesis from peach and grape fruits. Journal of Plant Physiology, 2009, 166(12): 1241-1252. doi: 10.1016/j.jplph.2009.01.013
[22]	HUBBARD K E, NISHIMURA N, HITOMI K, GETZOFF E D, SCHROEDER J I. Early abscisic acid signal transduction mechanisms: Newly discovered components and newly emerging questions. Genes & Development, 2010, 24(16): 1695-1708. doi: 10.1101/gad.1953910
[23]	BONEH U, BITON I, ZHENG C L, SCHWARTZ A, BEN-ARI G. Characterization of potential ABA receptors in Vitis vinifera. Plant Cell Reports, 2012, 31(2): 311-321. doi: 10.1007/s00299-011-1166-z
[24]	BONEH U, BITON I, SCHWARTZ A, BEN-ARI G. Characterization of the ABA signal transduction pathway in Vitis vinifera. Plant Science, 2012, 187: 89-96. doi: 10.1016/j.plantsci.2012.01.015
[25]	SPARVOLI F, MARTIN C, SCIENZA A, GAVAZZI G, TONELLI C. Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.). Plant Molecular Biology, 1994, 24(5): 743-755. doi: 10.1007/BF00029856
[26]	IBRAHIM R K, BRUNEAU A, BANTIGNIES B. Plant O- methyltransferases: molecular analysis, common signature and classification. Plant Molecular Biology, 1998, 36(1): 1-10. doi: 10.1023/A:1005939803300
[27]	NAKAYAMA T, SUZUKI H, NISHINO T. Anthocyanin acyltransferases: specificities, mechanism, phylogenetics, and applications. Journal of Molecular Catalysis B Enzymatic, 2003, 23(2): 117-132. doi: 10.1016/S1381-1177(03)00078-X
[28]	CONN S, CURTIN C, BEZIER A, FRANCO C, ZHANG W. Purification, molecular cloning, and characterization of glutathione S-transferases (GSTs) from pigmented Vitis vinifera L. cell suspension cultures as putative anthocyanin transport proteins. Journal of Experimental Botany, 2008, 59(13): 3621-3634. doi: 10.1093/jxb/ern217
[29]	PÉREZ-DÍAZ R, RYNGAJLLO M, PÉREZ-DÍAZ J, PEÑA-CORTÉS H, CASARETTO J A, GONZÁLEZ-VILLANUEVA E, RUIZ-LARA S. VvMATE1 and VvMATE2 encode putative proanthocyanidin transporters expressed during berry development in Vitis vinifera L. Plant Cell Reports, 2014, 33(7): 1147-1159. doi: 10.1007/s00299-014-1604-9
[30]	ZHAO J. Flavonoid transport mechanisms: How to go, and with whom. Trends in Plant Science, 2015, 20(9): 576-585. doi: 10.1016/j.tplants.2015.06.007
[31]	RINALDO A R, CAVALLINI E, JIA Y, MOSS S M A, MCDAVID D A J, HOOPER L C, ROBINSON S P, TORNIELLI G B, ZENONI S, FORD C M, BOSS P K, WALKER A R. A grapevine anthocyanin acyltransferase, transcriptionally regulated by VvMYBA, can produce most acylated anthocyanins present in grape skins. Plant Physiology, 2015, 169(3): 1897-1916.
[32]	牛铁泉, 董燕梅, 刘海霞, 张小军, 高燕, 张鹏飞, 梁长梅, 温鹏飞. 葡萄果实MYBA1与UFGT、DFR的作用机制. 中国农业科学, 2018, 51(12): 2368-2377.
	NIU T Q, DONG Y M, LIU H X, ZHANG X J, GAO Y, ZHANG P F, LIANG C M, WEN P F. The regulations of the MYBA1 in UFGT and DFR from the grape berries. Scientia Agricultura Sinica, 2018, 51(12): 2368-2377. (in Chinese)
[33]	FOURNIER-LEVEL A, LE CUNFF L, GOMEZ C, DOLIGEZ A, AGEORGES A, ROUX C, BERTRAND Y, SOUQUET J M, CHEYNIER V, THIS P. Quantitative genetic bases of anthocyanin variation in grape (Vitis vinifera L. ssp. sativa) berry: A quantitative trait locus to quantitative trait nucleotide integrated study. Genetics, 2009, 183(3): 1127-1139. doi: 10.1534/genetics.109.103929
[34]	MATUS J T, LOYOLA R, VEGA A, PEÑA-NEIRA A, BORDEU E, ARCE-JOHNSON P, ALCALDE J A. Post-veraison sunlight exposure induces MYB-mediated transcriptional regulation of anthocyanin and flavonol synthesis in berry skins of Vitis vinifera. Journal of Experimental Botany, 2009, 60(3): 853-867. doi: 10.1093/jxb/ern336
[35]	IMENE H, SIMON C, JEREMY P, CELINE L, STEFAN C, SERGE D, VIRGINIE L, JOCHEN B. The basic Helix-Loop-Helix transcription factor MYC1 is involved in the regulation of the flavonoid biosynthesis pathway in grapevine. Molecular Plant, 2010(3): 509-523.
[36]	XIE S, QIAO X L, CHEN H W, NAN H, ZHANG Z W. Coordinated regulation of grape berry flesh color by transcriptional activators and repressors. Journal of Agricultural and Food Chemistry, 2019, 67(42): 11815-11824. doi: 10.1021/acs.jafc.9b05234
[37]	PÉREZ-DÍAZ J R, PÉREZ-DÍAZ J, MADRID-ESPINOZA J, GONZÁLEZ-VILLANUEVA E, MORENO Y, RUIZ-LARA S. New member of the R2R3-MYB transcription factors family in grapevine suppresses the anthocyanin accumulation in the flowers of transgenic tobacco. Plant Molecular Biology, 2016, 90(1): 63-76. doi: 10.1007/s11103-015-0394-y
[38]	ALBERT N W, DAVIES K M, LEWIS D H, ZHANG H B, MONTEFIORI M, BRENDOLISE C, BOASE M R, NGO H, JAMESON P E, SCHWINN K E. A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots. The Plant Cell, 2014, 26(3): 962-980. doi: 10.1105/tpc.113.122069
[39]	ZHOU H, KUI L W, WANG F R, ESPLEY R V, REN F, ZHAO J B, OGUTU C, HE H P, JIANG Q, ALLAN A C, HAN Y P. Activator-type R2R3-MYB genes induce a repressor-type R2R3-MYB gene to balance anthocyanin and proanthocyanidin accumulation. The New Phytologist, 2019, 221(4): 1919-1934. doi: 10.1111/nph.2019.221.issue-4
[40]	ALBERT N W. Subspecialization of R2R3-MYB repressors for anthocyanin and proanthocyanidin regulation in forage legumes. Frontiers in Plant Science, 2015, 6: 1165.
[41]	GU R, LIU X F, ZHAO W S, YAN S S, SUN L H, WU B N, ZHANG X L. Functional characterization of the promoter and second intron of CUM1 during flower development in cucumber (Cucumis sativus L.). Horticultural Plant Journal, 2018(3): 103-110.
[42]	程寅胜, 陈健秋, 陈丹, 吕佳红, 张俊, 张绍铃, 伍涛, 张虎平. 梨糖转运相关基因PbTMT4启动子克隆及功能分析. 园艺学报, 2019, 46(1): 25-36.
	CHENG Y S, CHEN J Q, CHEN D, LÜ J H, ZHANG J, ZHANG S L, WU T, ZHANG H P. Cloning and functional analysis of the promoter of PbTMT4 gene related sugar transport in pear. Acta Horticulturae Sinica, 2019, 46(1): 25-36. (in Chinese)
[43]	FUJITA Y, FUJITA M, SATOH R, MARUYAMA K, PARVEZ M M, SEKI M, HIRATSU K, OHME-TAKAGI M, SHINOZAKI K, YAMAGUCHI-SHINOZAKI K. AREB1 is a transcription activator of novel ABRE-dependent ABA signaling that enhances drought stress tolerance in Arabidopsis. The Plant Cell, 2005, 17(12): 3470-3488. doi: 10.1105/tpc.105.035659
[44]	JEONG S T, GOTO-YAMAMOTO N, KOBAYASHI S, ESAKA M. Effects of plant hormones and shading on the accumulation of anthocyanins and the expression of anthocyanin biosynthetic genes in grape berry skins. Plant Science, 2004, 167(2): 247-252. doi: 10.1016/j.plantsci.2004.03.021
[45]	ZHAI X W, ZHANG Y S, KAI W B, LIANG B, JIANG L, DU Y W, WANG J A, SUN Y F, LENG P. Variable responses of two VlMYBA gene promoters to ABA and ACC in Kyoho grape berries. Journal of Plant Physiology, 2017, 211: 81-89. doi: 10.1016/j.jplph.2016.12.013

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
VvPAL	GTGAGGGAAGAACTGGGAGC	AATGAGCTTCCCTGCACACA	LOC100233012
VvCHS	CCGAGAAGTTACGCTCCACA	CTTCCTCAGCCGACTTCCTC	LOC100258106
VvC4H	GCCCATTTCTCCCAATATAGCAC	GTATGGAGAGAGGCCCTGGA	LOC100267215
VvF3H	CAAGGTGGCCTACAACGACT	AGGCCTCCACGATTTTCCTG	LOC100233079
VvUFGT	GAGGTCCTAGCACATGAGGC	CCTCCCATTGAGCCTTTGGT	LOC100233099
VvFAOMT	CAAAACTGAAGCCCTCACAAAGT	TCGACAGGGACATTCATCAGG	LOC100233134
VvGST	TGGAGGTTGAATCCCAACAATATCA	ACACATCCAGAACCTTACCCA	LOC100242506
VvMATE	AATTTTAATGGGTGGGAGGCCA	GAGAGACTGAGAGACTGCGAC	LOC 100250967
VvMYB4	ACAGCACTGGCGTCAAGAAT	TCTGCGACTGCTGGGAAATC	LOC100254518
VvNAC29	GCCCCGTATCCATTATCCCC	GTAGCTCGGTTGGGTCTAGC	LOC104879728
VvDof5.6	ATGCTCACTTGCTCCAGACC	TACCTTGGCTGAGAGAGGCT	LOC100265549
VvHD-ZIP	CCTGCAAAGAGGATCACCAT	GGTGCAATTCCCCGTAGTTA	LOC100245524
VvActin	TACAATTCCATCATGAAGTGTGATG	TTAGAAGCACTTCCTGTGAACAATG	LOC100246825

成分 Component	保留时间 Retention time (min)	分子/碎片 Molecular/Fragmention (m/z)	对照 Control (mg∙kg^-1)	ABA处理 ABA treatment (mg∙kg^-1)
飞燕草素 3-O-（6''-p-香豆酰葡萄糖苷） Delphinidin 3-O-(6''-p-coumaroyl-glucoside)	3.02	465.10/303.05	1.24±0.19	1.23±0.25
矮牵牛素 3-O-葡萄糖苷 Petunidin 3-O-glucoside	4.18	479.12/301.07	0.00	1.5±0.20**
芍药素 3-O-葡萄糖苷 Peonidin 3-O-glucoside	4.76	463.12/301.07	1.46±0.25	18.08±1.95**
锦葵色素 3-O-葡萄糖苷 Malvidin 3-O-glucoside	5.02	493.13/331.08	1.31±0.30	11.82±1.59**
天竺葵素 3-O-（6''-p-香豆酰葡萄糖苷） Cyanidin 3-O-(6''-p-coumaroyl-glucoside)	8.97	595.15/287.05	0.00	1.31±0.13**
芍药素 3-O-（6''-p-香豆酰葡萄糖苷） Peonidin 3-O-(6''-p-coumaroyl-glucoside)	9.94	639.17/317.06	0.00	3.72±0.25**
总花青苷 Total anthocyanins	/	/	4.23±0.28	37.5±2.36**

样本 Sample	原始序列 Number of raw reads	干净序列 Number of clean reads	有效比例 Effective reads ratio (%)	比对序列 Mapped reads (Mapping rate,%)	Q30值 Q30 (%)
ABA18h-rep1	46698448	45502062	97.44%	42661098 (93.76)	94.55
ABA 18h-rep2	42356456	41455998	97.87%	38818882 (93.64)	94.56
ABA 18h-rep3	47460348	46189130	97.32%	43310745 (93.77)	94.84
CK 18h-rep1	45626112	44517058	97.57%	41826560 (93.96)	94.73
CK 18h-rep2	46629670	44922268	96.34%	42176033 (93.89)	95.04
CK 18h-rep3	46173614	44415392	96.19%	41599095 (93.66)	94.91
ABA 3d-rep1	48528740	47335738	97.54%	44167501 (93.31)	95.09
ABA 3d-rep2	45802840	44487648	97.13%	41636081 (93.59)	95.33
ABA 3d-rep3	46843568	45789804	97.75%	42741325 (93.34)	94.74
CK 3d-rep1	41352252	40054402	96.86%	36726263 (91.69)	94.33
CK 3d-rep2	46588212	45041810	96.68%	41795220 (92.79)	94.3
CK 3d-rep3	46096744	44484560	96.50%	40918000 (91.98)	94.44