水稻WRKY转录因子基因家族响应外源一氧化氮的表达谱分析

doi:10.16819/j.1001-7216.2016.5138

摘要/Abstract

摘要：

一氧化氮(nitric oxide, NO)信号分子与WRKY转录因子均参与植物抗逆、发育与代谢等许多生理过程。采用Agilent水稻全基因组cDNA芯片分析了NO处理后1、6和12 h水稻幼苗WRKY转录因子基因的表达谱,鉴定出在1个时间点有两倍或两倍以上表达变化的WRKY基因32个,主要分布在WRKY的Ⅰ和Ⅱ组,其中75%的Ⅱa和45.6%的Ⅱd亚组成员为差异表达基因;鉴定出至少在2个时间点有两倍或两倍以上表达变化的WRKY基因15个,均为早期(1 h)应答,且多数(64.2%)持续上调;基因功能预测分析表明,这些基因主要参与生物学过程中的细胞过程、代谢过程和刺激响应,以及分子功能中的转录调节活性和结合;代谢通路分析表明,WRKY24涉及植物与病原菌相互作用代谢通路。实时荧光定量PCR 验证结果与芯片杂交结果基本一致,印证了芯片杂交结果的有效性。上述发现提示,NO信号可能参与了WRKY转录因子介导的生物学调控功能,并为这些基因的进一步功能分析奠定基础。

关键词: 水稻, WRKY转录因子, 一氧化氮, 基因表达谱

Abstract:

Signal molecule nitric oxide (NO) and WRKY transcription factors are involved in many physiological processes, such as defense against stresses, development and metabolism in plants. In the present study, we investigated the expression patterns of WRKY transcription factor gene family in rice seedlings at 1, 6 and 12 after exogenous NO treatments using Agilent rice cDNA oligo microarray. Totally, 32 differently expressed WRKY genes, whose expression levels increased or decreased by at least two-fold at one time point compared with the control (T0), were identified. These WRKY genes were mainly distributed over Groups Ⅰ and Ⅱ, among which 75% of Ⅱa and 45.6% of Ⅱd subgroup members were differently expressed upon the NO treatments. Moreover, 15 identified NO-responsive WRKY genes, whose expression level changed by more than two-fold at least two time points among the three time points compared with the control, exhibited an earlier (1 h) response to the NO treatments, and most of them (64.2%) were continually up-regulated. Prediction of gene function revealed that the NO-responsive WRKY genes were mainly involved in cellular process, metabolic process and response to stimulus of biological process, and transcription regulator activity and binding of molecular function. The analysis of metabolic pathways showed that WRKY24 was involved in plant-pathogen interaction pathway. The results of microarray hybridization were largely consistent with those of quantitative real-time PCR, verifying the validity of microarray hybridization. These findings suggest that NO signaling might be involved in the regulatory functions of WRKY transcription factors, and provide a basis for further functional research for these differentially expressed WRKY genes.

Key words: rice (Oryza sativa), WRKY transcription factor, nitric oxide, gene expression profile

中图分类号:

Q755
S511.01

孟姣, 王海华, 向建华, 蒋丹, 彭喜旭, 贺欢欢. 水稻WRKY转录因子基因家族响应外源一氧化氮的表达谱分析[J]. 中国水稻科学, 2016, 30(2): 111-120.

Jiao MENG, Hai-hua WANG, Jian-hua XIANG, Dan JIANG, Xi-xu PENG, Huan-huan HE. Expression Profiles of Rice WRKY Transcription Factor Gene Family Responsive to Exogenous Nitric Oxide Application[J]. Chinese Journal OF Rice Science, 2016, 30(2): 111-120.

图/表 9

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基因 Gene	登录号 Accession No.	正向引物 Forward primer (5'-3')	反向引物 Reverse primer (5'-3')
OsWRKY15	Os01g0656400	GTCGTCGCCCTTCGTGTC	ATGAACTCCATGTCGTCTCCC
OsWRKY42	Os02g0462800	ATGGGAGCGTGTCCAACG	GCGGCCATCAGAAGCATAA
OsWRKY21	Os01g0821600	GCTGAGCAACAGAGCAGGTA	GCGTCAGTTATGCGTCAAG
OsWRKY77	Os01g0584900	CCTCGCTGCTGCTTCCTT	TTCAGGTCGCCTTGGTCC
OsWRKY71	Os02g0181300	AGGAATTGATGATTCGCTGTA	CGCCGATGATCGTTGGTT
OsWRKY24	Os01g0826400	TGCTCCTGACCTCCAGTATCTT	TTCCTCTGCTCGTCCTTGC
OsWRKY69	Os08g0386200	TGCTTCGACTTCGACCCG	TCTTCCCTCCTGCCACCA
Actin 1	Os03g0718100	ATCGCCCTGGACTATGAC	GAAACGCTCAGCACCAAT

基因 Gene	登录号 Accession No.	正向引物 Forward primer (5'-3')	反向引物 Reverse primer (5'-3')
OsWRKY15	Os01g0656400	GTCGTCGCCCTTCGTGTC	ATGAACTCCATGTCGTCTCCC
OsWRKY42	Os02g0462800	ATGGGAGCGTGTCCAACG	GCGGCCATCAGAAGCATAA
OsWRKY21	Os01g0821600	GCTGAGCAACAGAGCAGGTA	GCGTCAGTTATGCGTCAAG
OsWRKY77	Os01g0584900	CCTCGCTGCTGCTTCCTT	TTCAGGTCGCCTTGGTCC
OsWRKY71	Os02g0181300	AGGAATTGATGATTCGCTGTA	CGCCGATGATCGTTGGTT
OsWRKY24	Os01g0826400	TGCTCCTGACCTCCAGTATCTT	TTCCTCTGCTCGTCCTTGC
OsWRKY69	Os08g0386200	TGCTTCGACTTCGACCCG	TCTTCCCTCCTGCCACCA
Actin 1	Os03g0718100	ATCGCCCTGGACTATGAC	GAAACGCTCAGCACCAAT

基因 Gene	探针 Probe	表达倍数 Expression fold change			组别 Group
基因 Gene	探针 Probe	T1/T0		T6/T0	T12/T0
OsWRKY1	Os01g0246700\|mRNA\|AY676928\|CDS	1.87	2.06	1.35	Ⅱb
	Os01g0246700\|mRNA\|AK105509\|CDS+3'UTR	2.72	1.54	2.19
OsWRKY3	Os03g0758000\|mRNA\|AY341859\|CDS+3'UTR	0.33	0.38	3.70	Ⅰb
OsWRKY6	Os03g0798500\|COMBINER_EST\|Os03g0798500\|8	0.59	0.57	2.25	Ⅱc
OsWRKY8	Os05g0583000\|mRNA\|AK109568\|CDS+3'UTR	2.95	1.13	0.44	Ⅱd
OsWRKY11	Os01g0626400\|mRNA\|AY341856\|CDS+3'UTR	1.91	2.88	1.60	Ⅰb
	Os01g0626400\|mRNA\|AK108745\|CDS+3'UTR	2.23	2.57	1.63
OsWRKY12	Os01g0624700\|mRNA\|AK111416\|CDS+3'UTR	3.14	2.74	1.99	Ⅱd
OsWRKY13	Os01g0750100\|mRNA\|AK060018\|CDS+3'UTR	1.55	0.67	0.48	Ⅱd
OsWRKY15	Os01g0656400\|mRNA\|AY676926\|CDS	4.98	1.16	2.70	Ⅲa
OsWRKY17	Os01g0972800\|mRNA\|AY341842\|CDS+3'UTR	0.09	0.09	0.32	Ⅰb
OsWRKY21	Os01g0821600\|mRNA\|AK108657\|CDS+3'UTR	4.69	4.42	1.31	Ⅲb
OsWRKY23	Os01g0734000\|mRNA\|AK108909\|CDS+3'UTR	2.24	1.69	2.11	Ⅰb
OsWRKY24	Os01g0826400\|mRNA\|AK107199\|CDS+3'UTR	6.48	0.83	3.86	Ⅰa
	Os01g0826400\|mRNA\|AY341849\|CDS+3'UTR	5.39	0.63	3.33
	Os01g0826400\|mRNA\|AY676925\|CDS	5.92	0.75	3.65
OsWRKY28	Os06g0649000\|mRNA\|AK106282\|CDS+3'UTR	7.87	1.32	2.28	Ⅱa
	Os06g0649000\|mRNA\|AK119644\|CDS+3'UTR	6.77	1.05	1.91
OsWRKY29	Os07g0111400\|mRNA\|AY341858\|CDS+3'UTR	0.73	1.32	2.70	Ⅰb
OsWRKY36	Os04g0545000\|mRNA\|AK073695\|CDS+3'UTR	0.47	0.73	0.61	Ⅰb
OsWRKY39	Os02g0265200\|mRNA\|AK066775\|CDS+3'UTR	1.43	2.00	2.62	Ⅱd
OsWRKY42	Os02g0265200\|mRNA\|AK119593\|CDS+3'UTR	1.42	2.27	2.34	Ⅱc
	Os02g0462800\|mRNA\|AK110587\|CDS+3'UTR	5.51	3.80	2.01
	Os02g0462800\|mRNA\|AY676923\|CDS	5.01	3.14	2.05
OsWRKY50	Os11g0117600\|COMBINER_EST\|Os11g0117600\|8	0.65	7.31	1.11	Ⅲb
OsWRKY53	Os05g0343400\|mRNA\|AY676929\|CDS	2.10	0.89	1.02	Ⅰa
OsWRKY56	Os12g0116600\|mRNA\|AK102093\|CDS+3'UTR	1.31	0.42	1.03	Ⅲb
OsWRKY60	Os03g0657400\|COMBINER_EST\|AU057193\|6	1.01	0.58	0.44	Ⅰb
OsWRKY62	Os09g0417800\|mRNA\|AK067834\|CDS+3'UTR	1.48	1.65	2.03	Ⅱa
OsWRKY67	Os05g0183100\|mRNA\|AK066252\|CDS+3'UTR	1.13	0.66	0.28	Ⅰb
OsWRKY69	Os08g0386200\|mRNA\|AK111606\|CDS+3'UTR	3.05	0.98	2.77	Ⅲa
OsWRKY71	Os08g0386200\|mRNA\|AY676931\|CDS	3.33	0.88	2.80	Ⅱa
	Os02g0181300\|mRNA\|AK058773\|CDS+3'UTR	3.79	1.72	1.67
	Os02g0181300\|mRNA\|AY676927\|CDS	3.41	1.45	1.64
OsWRKY72	Os11g0490900\|mRNA\|AK108860\|CDS+3'UTR	2.32	0.89	1.75	Ⅰb
OsWRKY77	Os01g0584900\|mRNA\|AK108522\|CDS+3'UTR	1.10	1.80	2.31	Ⅰb
	Os01g0584900\|mRNA\|AY341846\|CDS+3'UTR	1.24	1.88	2.48
OsWRKY79	Os03g0335200\|mRNA\|AK105244\|CDS+3'UTR	0.66	1.83	2.53	Ⅲb
OsWRKY88	Os07g0596900\|mRNA\|AY341847\|CDS+3'UTR	0.94	1.53	2.13	Ⅲa
OsWRKY104	Os11g0117400\|COMBINER_EST\|CI444468\|6	1.40	0.32	0.93	Ⅲb
OsWRKY109	Os05g0129800\|COMBINER_EST\|Os05g0129800\|8	0.73	0.82	2.02	Ⅱd
OsWRKY113	Os06g0158100\|mRNA\|AY676932\|CDS	2.35	0.96	3.71	Ⅲa

基因 Gene	探针 Probe	表达倍数 Expression fold change			组别 Group
基因 Gene	探针 Probe	T1/T0		T6/T0	T12/T0
OsWRKY1	Os01g0246700\|mRNA\|AY676928\|CDS	1.87	2.06	1.35	Ⅱb
	Os01g0246700\|mRNA\|AK105509\|CDS+3'UTR	2.72	1.54	2.19
OsWRKY3	Os03g0758000\|mRNA\|AY341859\|CDS+3'UTR	0.33	0.38	3.70	Ⅰb
OsWRKY6	Os03g0798500\|COMBINER_EST\|Os03g0798500\|8	0.59	0.57	2.25	Ⅱc
OsWRKY8	Os05g0583000\|mRNA\|AK109568\|CDS+3'UTR	2.95	1.13	0.44	Ⅱd
OsWRKY11	Os01g0626400\|mRNA\|AY341856\|CDS+3'UTR	1.91	2.88	1.60	Ⅰb
	Os01g0626400\|mRNA\|AK108745\|CDS+3'UTR	2.23	2.57	1.63
OsWRKY12	Os01g0624700\|mRNA\|AK111416\|CDS+3'UTR	3.14	2.74	1.99	Ⅱd
OsWRKY13	Os01g0750100\|mRNA\|AK060018\|CDS+3'UTR	1.55	0.67	0.48	Ⅱd
OsWRKY15	Os01g0656400\|mRNA\|AY676926\|CDS	4.98	1.16	2.70	Ⅲa
OsWRKY17	Os01g0972800\|mRNA\|AY341842\|CDS+3'UTR	0.09	0.09	0.32	Ⅰb
OsWRKY21	Os01g0821600\|mRNA\|AK108657\|CDS+3'UTR	4.69	4.42	1.31	Ⅲb
OsWRKY23	Os01g0734000\|mRNA\|AK108909\|CDS+3'UTR	2.24	1.69	2.11	Ⅰb
OsWRKY24	Os01g0826400\|mRNA\|AK107199\|CDS+3'UTR	6.48	0.83	3.86	Ⅰa
	Os01g0826400\|mRNA\|AY341849\|CDS+3'UTR	5.39	0.63	3.33
	Os01g0826400\|mRNA\|AY676925\|CDS	5.92	0.75	3.65
OsWRKY28	Os06g0649000\|mRNA\|AK106282\|CDS+3'UTR	7.87	1.32	2.28	Ⅱa
	Os06g0649000\|mRNA\|AK119644\|CDS+3'UTR	6.77	1.05	1.91
OsWRKY29	Os07g0111400\|mRNA\|AY341858\|CDS+3'UTR	0.73	1.32	2.70	Ⅰb
OsWRKY36	Os04g0545000\|mRNA\|AK073695\|CDS+3'UTR	0.47	0.73	0.61	Ⅰb
OsWRKY39	Os02g0265200\|mRNA\|AK066775\|CDS+3'UTR	1.43	2.00	2.62	Ⅱd
OsWRKY42	Os02g0265200\|mRNA\|AK119593\|CDS+3'UTR	1.42	2.27	2.34	Ⅱc
	Os02g0462800\|mRNA\|AK110587\|CDS+3'UTR	5.51	3.80	2.01
	Os02g0462800\|mRNA\|AY676923\|CDS	5.01	3.14	2.05
OsWRKY50	Os11g0117600\|COMBINER_EST\|Os11g0117600\|8	0.65	7.31	1.11	Ⅲb
OsWRKY53	Os05g0343400\|mRNA\|AY676929\|CDS	2.10	0.89	1.02	Ⅰa
OsWRKY56	Os12g0116600\|mRNA\|AK102093\|CDS+3'UTR	1.31	0.42	1.03	Ⅲb
OsWRKY60	Os03g0657400\|COMBINER_EST\|AU057193\|6	1.01	0.58	0.44	Ⅰb
OsWRKY62	Os09g0417800\|mRNA\|AK067834\|CDS+3'UTR	1.48	1.65	2.03	Ⅱa
OsWRKY67	Os05g0183100\|mRNA\|AK066252\|CDS+3'UTR	1.13	0.66	0.28	Ⅰb
OsWRKY69	Os08g0386200\|mRNA\|AK111606\|CDS+3'UTR	3.05	0.98	2.77	Ⅲa
OsWRKY71	Os08g0386200\|mRNA\|AY676931\|CDS	3.33	0.88	2.80	Ⅱa
	Os02g0181300\|mRNA\|AK058773\|CDS+3'UTR	3.79	1.72	1.67
	Os02g0181300\|mRNA\|AY676927\|CDS	3.41	1.45	1.64
OsWRKY72	Os11g0490900\|mRNA\|AK108860\|CDS+3'UTR	2.32	0.89	1.75	Ⅰb
OsWRKY77	Os01g0584900\|mRNA\|AK108522\|CDS+3'UTR	1.10	1.80	2.31	Ⅰb
	Os01g0584900\|mRNA\|AY341846\|CDS+3'UTR	1.24	1.88	2.48
OsWRKY79	Os03g0335200\|mRNA\|AK105244\|CDS+3'UTR	0.66	1.83	2.53	Ⅲb
OsWRKY88	Os07g0596900\|mRNA\|AY341847\|CDS+3'UTR	0.94	1.53	2.13	Ⅲa
OsWRKY104	Os11g0117400\|COMBINER_EST\|CI444468\|6	1.40	0.32	0.93	Ⅲb
OsWRKY109	Os05g0129800\|COMBINER_EST\|Os05g0129800\|8	0.73	0.82	2.02	Ⅱd
OsWRKY113	Os06g0158100\|mRNA\|AY676932\|CDS	2.35	0.96	3.71	Ⅲa