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.

图/表 13

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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

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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
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