Genome-wide Identification of WRKY Transcription Factors and Key Genes for Abscisic Acid-mediated Flavonoid Synthesis in Sour Jujube (Ziziphus jujuba var. spinosa)

doi:10.7525/j.issn.1673-5102.2026.05.007

Abstract

Abstract:

WRKY transcription factors play important roles in flavonoid biosynthesis and plant growth and development； however， their functional roles in the medicinal plant sour jujube （Ziziphus jujuba var. spinosa） remain largely unexplored. In this study， we conducted a comprehensive genome-wide identification of the WRKY gene family in sour jujube using bioinformatics approaches. We systematically analyzed their phylogenetic relationships， chromosomal localization， conserved domains， and cis-acting elements in promoter regions. Furthermore， we performed exogenous abscisic acid（ABA） treatment on sour jujube seeds， combined with flavonoid content determination and gene expression analysis， to screen candidate genes significantly associated with flavonoid accumulation. The tissue-specific expression patterns of these candidate genes were subsequently validated by quantitative real-time PCR. A total of 47 ZjWRKY genes were identified in the sour jujube genome， which were classified into three subfamilies（Group Ⅰ， Group Ⅱ， and Group Ⅲ） based on phylogenetic analysis. The ZjWRKY genes were unevenly distributed across 11 chromosomes， and all encoded proteins contained the conserved WRKYGQK domain， predominantly localized in the nucleus. Notably， 89% of ZjWRKY genes contained ABA-responsive elements（ABREs） within their promoter regions. Exogenous ABA treatment significantly promoted flavonoid accumulation in germinated sour jujube seeds， and the flavonoid content was correlated with the expression patterns of 12 ZjWRKY genes. Among them， ZjWRKY10， ZjWRKY30， and ZjWRKY45 showed a strong correlation with flavonoid content and were predominantly and highly expressed in seeds and pulp tissues. These results suggest that the three ZjWRKY genes may act synergistically in regulating fruit and seed development as well as ABA-induced flavonoid biosynthesis in sour jujube. This study provides important clues for further elucidating the ABA signaling-WRKY transcription factor-flavonoid biosynthesis regulatory network.

Key words: Ziziphus jujuba var. spinosa, WRKY transcription factors, abscisic acid, flavonoids, seed

CLC Number:

Q943.2

Chuxing DONG, Mengyuan ZHANG, Xinbo SHI, Gang ZHANG, Zhongxing SONG, Nan WANG, Honghong JIAO. Genome-wide Identification of WRKY Transcription Factors and Key Genes for Abscisic Acid-mediated Flavonoid Synthesis in Sour Jujube (Ziziphus jujuba var. spinosa)[J]. Bulletin of Botanical Research, 2026, 46(5): 851-865.

Figures/Tables 11

Table 1

Table 2

Physicochemical property analysis of ZjWRKY gene family proteins in Ziziphus jujuba

基因ID Gene ID	基因名称 Gene name	蛋白长度 Protein length/aa	相对分子质量 Relative molecular mass/kDa	等电点 pI	不稳定指数 Instability index	脂溶性系数 Aliphatic index	总疏水性 GRAVY	亚细胞定位 Subcellular localization
ZspiChr4G00070360.1	ZjWRKY1	501	55 053.62	6.40	44.30	58.64	-0.992	细胞核
ZspiChr2G00176960.1	ZjWRKY2	592	64 619.35	7.21	60.56	46.81	-0.928	细胞核
ZspiChr1G00015850.1	ZjWRKY3	521	57 263.14	5.12	63.06	59.52	-0.930	细胞核
ZspiChr12G00259750.1	ZjWRKY4	477	52 053.75	9.09	47.12	57.88	-0.895	细胞核
ZspiChr12G00265570.1	ZjWRKY5	373	41 018.42	5.16	70.12	50.75	-0.724	细胞核
ZspiChr10G00218060.1	ZjWRKY6	310	34 331.69	5.65	58.67	54.68	-0.949	细胞核
ZspiChr11G00191570.1	ZjWRKY7	184	21 092.86	9.20	42.74	71.52	-0.899	细胞核
ZspiChr12G00259570.1	ZjWRKY8	207	23 355.69	9.26	54.66	62.56	-0.771	过氧化物酶体
ZspiChr1G00006330.1	ZjWRKY9	336	37 387.94	6.37	52.64	46.46	-0.976	细胞核
ZspiChr4G00073720.1	ZjWRKY10	345	39 158.92	6.76	62.93	47.48	-1.101	细胞核
ZspiChr9G00139950.1	ZjWRKY11	195	22 341.12	9.34	36.81	50.51	-0.921	细胞核
ZspiChr8G00160490.1	ZjWRKY12	198	21 938.16	6.73	49.17	41.31	-0.934	细胞核
ZspiChr6G00036910.1	ZjWRKY13	173	19 750.96	5.59	49.56	57.40	-0.842	细胞外
ZspiChr5G00099850.1	ZjWRKY14	199	23 062.84	9.21	45.42	50.90	-1.012	细胞核
ZspiChr9G00117920.1	ZjWRKY15	335	37 135.37	6.43	58.37	58.00	-0.728	细胞核
ZspiChr10G00210320.1	ZjWRKY16	192	21 894.40	9.43	45.48	49.17	-0.961	细胞核
ZspiChr9G00131150.1	ZjWRKY17	503	55 917.06	5.50	49.71	60.70	-0.856	细胞核
ZspiChr8G00160700.1	ZjWRKY18	242	27 914.17	8.66	63.80	54.38	-0.973	细胞核
ZspiChr1G00002140.1	ZjWRKY19	328	35 300.64	9.57	56.26	56.19	-0.605	细胞核
ZspiChr11G00199490.1	ZjWRKY20	299	33 456.13	5.36	56.80	66.49	-0.764	细胞核
ZspiChr9G00117330.1	ZjWRKY21	507	55 356.96	7.41	51.06	65.11	-0.641	细胞核
ZspiChr8G00154750.1	ZjWRKY22	329	35 931.61	9.53	42.72	70.27	-0.568	细胞核
ZspiChr6G00041700.1	ZjWRKY23	366	39 473.46	9.64	50.56	60.27	-0.578	细胞核
ZspiChr6G00034290.1	ZjWRKY24	371	40 189.57	9.57	58.58	66.82	-0.558	细胞核
ZspiChr1G00028160.1	ZjWRKY25	316	35 076.43	8.68	45.86	64.84	-0.734	细胞核
ZspiChr9G00122400.1	ZjWRKY26	361	40 626.82	9.65	59.45	65.37	-0.855	细胞核
ZspiChr1G00005320.1	ZjWRKY27	312	34 716.29	5.60	56.82	43.43	-0.777	叶绿体
ZspiChr11G00191590.1	ZjWRKY28	392	44 753.87	4.89	67.54	63.60	-0.701	细胞核
ZspiChr12G00259500.1	ZjWRKY29	355	38 920.61	5.92	66.41	44.82	-0.774	细胞核
ZspiChr9G00118050.1	ZjWRKY30	289	32 228.5	5.27	53.06	50.59	-1.017	细胞核
ZspiChr8G00145050.1	ZjWRKY31	327	37 411.67	5.13	57.02	62.32	-0.758	细胞核
ZspiChr11G00190750.1	ZjWRKY32	370	41 879.58	5.27	62.82	58.22	-0.810	细胞核
ZspiChr3G00239020.1	ZjWRKY33	362	40 124.62	5.68	56.69	57.71	-0.777	细胞核
ZspiChr2G00185960.1	ZjWRKY34	738	80 905.86	5.90	52.26	60.14	-0.688	细胞核
ZspiChr10G00217520.1	ZjWRKY35	389	43 130.26	7.70	50.74	50.59	-1.050	细胞核
ZspiChr11G00202900.1	ZjWRKY36	1 258	138 887.95	8.23	47.52	84.80	-0.335	细胞核
ZspiChr1G00020420.1	ZjWRKY37	590	64 658.71	6.12	51.73	64.86	-0.703	细胞核
ZspiChr1G00010250.1	ZjWRKY38	758	81 979.08	5.57	52.18	57.56	-0.763	细胞核
ZspiChr1G00011820.1	ZjWRKY39	633	67 853.91	6.12	41.23	63.76	-0.633	细胞核
ZspiChr4G00067590.1	ZjWRKY40	227	24 273.76	4.77	69.68	64.93	-0.434	细胞核
ZspiChr2G00185940.1	ZjWRKY41	350	39 454.27	6.01	60.99	64.63	-0.649	高尔基体
ZspiChr11G00203510.1	ZjWRKY42	319	36 035.82	5.25	59.92	64.80	-0.762	细胞核
ZspiChr3G00243560.1	ZjWRKY43	391	43 607.65	5.90	50.63	52.61	-0.859	细胞核
ZspiChr4G00065800.1	ZjWRKY44	571	61 532.69	5.98	45.34	59.39	-0.768	细胞核
ZspiChr3G00239030.1	ZjWRKY45	321	36 025.77	5.27	47.54	59.22	-0.763	细胞核
ZspiChr11G00203440.1	ZjWRKY46	324	36 876.17	5.79	47.93	71.88	-0.687	细胞核
ZspiChr6G00038170.1	ZjWRKY47	316	34 698.02	8.64	42.63	72.97	-0.542	细胞核

Table 2

Fig.1

Fig.2

Fig.3

Fig.4

Table 3

Fig.5

Fig.6

Table 4

Fig.7

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基因名称 Gene name	正向引物序列（5′→3′） Forward primer sequence（5′→3′）	反向引物序列（5′→3′） Reverse primer sequence（5′→3′）
ZjH3	CTTGGTCGGTCTGTTCGAGG	ATGCCGGATCTAAGCCCTCT
ZjWRKY1	ATCCGAACTGCCTCGTGAAA	CATCTGGCCATGCTCATCCA
ZjWRKY2	GTGTTGAGGAGCGTACTGGT	CAGCCTGAGCTGCAAATGTT
ZjWRKY10	TCCAATGAAGCTCCTGCGAGAC	AGGGCTGTGTGTGTGTGTGT
ZjWRKY11	TAGTGCTGATCATGGGGCTG	GTTAGGCGCTGGACTTGCTT
ZjWRKY12	TGCTGCTACTGCCACATCAA	AGCTTGAGTCTTTTCCGTCCC
ZjWRKY24	ATGTCTCAGTCATCCGCTGG	TTTGATGGGTTTTTGGCCGT
ZjWRKY25	CAACCATCCTCACCCCTCAC	GAAATGTCGAACTTCCGGCG
ZjWRKY30	TCTCGCTGATCAACCACCAC	TCGGGAACACCTAATTGGGG
ZjWRKY31	GAGTCAGGGCTTGATGGACC	GGTATGCCGTCCTCGGTATG
ZjWRKY32	TTGGAACGCAGAGAGGTTGT	CCTGCAAGGTTTTTCATCCG
ZjWRKY36	CCTGTCTGCCTTGAGGTGTA	AGGAGGCGGAGAGAGAGGTA
ZjWRKY37	TGGGAATAACTTGTGCCGCT	ACAGCTTTGGGATGGACAACT
ZjWRKY44	TCTCCGCCATCTCTCTCTGTCTGA	ACCACTGATTGATCGCTGCT
ZjWRKY45	TCTGAGAATTCCGGCGAGAG	GCTCGGCAACCTTGATCGTA
ZjWRKY47	ATGAATTAGCTCCGCAGGCA	AACTGCACTCGGTGTTTCCA

基因对 Gene pairs	同义突变率 K_a	非同义突变率 K_s	同义突变率/非同义突变率 K_a/K_s	选择压力 Selection pressure
ZjWRKY9/ZjWRKY10	0.319	2.373	0.134	纯化选择
ZjWRKY40/ZjWRKY27	0.417	1.516	0.275	纯化选择
ZjWRKY44/ZjWRKY39	0.287	3.056	0.094	纯化选择
ZjWRKY25/ZjWRKY47	0.362	NaN	NaN	无法计算
ZjWRKY2/ZjWRKY36	0.319	1.991	0.160	纯化选择
ZjWRKY43/ZjWRKY32	0.381	NaN	NaN	无法计算
ZjWRKY43/ZjWRKY31	0.340	2.847	0.126	纯化选择
ZjWRKY31/ZjWRKY32	0.415	2.461	0.169	纯化选择
ZjWRKY14/ZjWRKY11	0.370	1.581	0.234	纯化选择
ZjWRKY22/ZjWRKY24	0.216	1.987	0.108	纯化选择

基因名称 Gene name	基因ID Gene ID	ABA 24 vs CK		ABA 48 vs CK
基因名称 Gene name	基因ID Gene ID	log₂（FoldChange）	P_adj	log₂（FoldChange）	P_adj
CHI	ZspiChr1G00028950	1.231 96	<0.001	-0.488 99	<0.001
CHI	ZspiChr5G00109140	2.773 76	<0.001	1.451 71	<0.001
F3H	ZspiChr5G00095680	0.978 29	<0.001	1.069 25	<0.001
CHS	ZspiChr9G00134850	-2.898 93	<0.001	-2.688 15	<0.001
FLS	ZspiChr1G00006420	-1.007 06	<0.001	-0.641 72	<0.001
CCR	ZspiChr1G00008930	-0.744 16	<0.001	2.402 25	<0.001
CCR	ZspiChr6G00030760	0.000 02	<0.001	1.662 04	<0.001
ANR	ZspiChr11G00190780	1.480 21	<0.001	1.885 05	<0.001
ANR	ZspiChr1G00018920	1.501 35	<0.001	1.024 85	<0.001