茶树CsWRKYIIcs转录因子的克隆及功能分析

doi:10.3864/j.issn.0578-1752.2020.12.013

摘要/Abstract

摘要：

【目的】以‘陕茶1号’为材料,克隆CsWRKYIIcs转录因子并分析序列特征,了解其在不同组织和非生物胁迫下的表达模式以及转录活性,为深入研究茶树在逆境胁迫中的功能提供理论依据。【方法】根据茶树基因组数据库注释的WRKY序列设计特异性引物,采用RT-PCR技术从‘陕茶1号’中扩增CsWRKYIIcs的cDNA序列,用生物信息工具分析序列的特点,实时荧光定量PCR（qRT-PCR）研究基因的表达模式,用酵母试验验证其转录活性。【结果】获得9条CsWRKYIIcs的cDNA序列,开放阅读框长度分别为561、960、936、978、897、912、720、1 008和969 bp,分别编码186、319、311、325、298、303、239、335、322个氨基酸。除了CsWRKYIIc7缺少锌指序列外,其他CsWRKYIIcs均含有1个WRKY保守结构域和典型的C₂H₂型锌指结构。不同物种中的WRKYIIcs具有相似的保守基序,而茶树CsWRKYIIcs和拟南芥、葡萄等双子叶植物的WRKYIIcs氨基酸序列相似性更高。另外,CsWRKYIIcs启动子区域均预测到多个与非生物胁迫相关的顺式作用元件,意味着其可能参与非生物胁迫响应。qRT-PCR结果表明,9个CsWRKYIIcs在根和花中的表达量高于茎和叶,具有一定的特异性。同时,在干旱、ABA、高温和高盐胁迫下被不同程度的诱导表达,其中CsWRKYIIc1和CsWRKYIIc7的表达量变化显著,与推定的顺式作用元件结果相符。酵母试验表明9个茶树CsWRKYIIcs均具有转录激活活性。【结论】克隆获得9个茶树CsWRKYIIcs转录因子,它们参与了茶树对ABA、干旱、高温和高盐胁迫的响应,也可能发挥转录激活因子的调控作用。CsWRKYIIc1和CsWRKYIIc7可作为候选基因深入研究茶树的抗逆功能。

关键词: 茶树, CsWRKYIIcs, 克隆, 表达模式, 转录活性

Abstract:

【Objective】 The objective of this study was to clone CsWRKYIIcs transcription factors from the tea plant (Camellia sinensis) ‘Shaancha No.1’ and analyze their sequence characteristics, to investigate the expression patterns in different tissues and under abiotic stresses, and to verify the transcription activity, thus providing a basis for further exploring the functions of tea plants under abiotic stresses. 【Method】 Specific primers were designed based on the annotated WRKY sequences released in tea genome database. RT-PCR was used to amplify the cDNA sequences of CsWRKYIIcs from the tea plant ‘Shaancha No.1’, the bioinformatical tools were carried out to analyze the sequence characteristics, and the real-time fluorescence quantification PCR (qRT-PCR) was employed to investigate the expression patterns. Y2H assay was applied to verify the transcription activity. 【Result】 Nine cDNA sequences of the CsWRKYIIcs were obtained with the open reading frame length of 561, 960, 936, 978, 897, 912, 720, 1008 and 969 bp, encoding 186, 319, 311, 325, 298, 303, 239, 239, 335 and 322 amino acids, respectively. Except for the CsWRKYIIc7 which lacked zinc finger sequences, each of all the other CsWRKYIIcs contained one conserved WRKY domain and a typical C₂H₂-type zinc finger motif. WRKYIIcs had similar conserved motifs in different species, and CsWRKYIIcs from the tea plant showed higher identity at the amino acids level with those from dicotyledonous Arabidopsis thaliana and Vitis vinifera. Furthermore, multiple cis-elements related to abiotic stresses were predicted in the promoter regions, implying that the CsWRKYIIcs might involve in response to various abiotic stresses. qRT-PCR test results suggested that the expression patterns of nine CsWRKYIIcs in different tissues were quite specific, with higher expression level in roots and flowers than that in stems and leaves. Meanwhile, the nine CsWRKYIIcs showed different expression patterns when induced by drought, ABA, high temperature and high salinity stress; the expression of CsWRKYIIc1 and CsWRKYIIc7 changed most significantly, which were consistent with the result of putative cis-elements. In addition, the Y2H assay results indicated that all the nine CsWRKYIIcs had transcriptional activation activity. 【Conclusion】 Nine CsWRKYIIcs transcription factors cloned from the tea plant were involved in response to ABA, drought, high temperature and high salinity stress, and they might play a regulatory role of transcriptional activators. CsWRKYIIc1 and CsWRKYIIc7 might be used as candidate genes for further research into the anti-adversity function of tea plants.

Key words: Camellia sinensis, CsWRKYIIcs, cloning, expression patterns, transcription activity

肖罗丹, 唐磊, 王伟东, 高岳芳, 黄伊凡, 孟阳, 杨亚军, 肖斌. 茶树CsWRKYIIcs转录因子的克隆及功能分析[J]. 中国农业科学, 2020, 53(12): 2460-2476.

XIAO LuoDan, TANG Lei, WANG WeiDong, GAO YueFang, HUANG YiFan, MENG Yang, YANG YaJun, XIAO Bin. Cloning and Functional Analysis of CsWRKYIIcs Transcription Factors in Tea Plant[J]. Scientia Agricultura Sinica, 2020, 53(12): 2460-2476.

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图/表 14

表1

克隆、荧光定量和构建酵母载体引物"

引物 Primer	上游序列（5′-3′） Upstream primer	下游序列（5′-3′） Downstream primer	作用 Function
CsWRKYIIc1	ATC CTT CCA AAC GAT GAC ATT	ACCATTGTAAGTTGTACACATGG	克隆 Cloning
CsWRKYIIc2	TGAAGAAGAATCTGTGCTCCT	TCTAAAGATAACCCAACAAACCTC
CsWRKYIIc3	AGAAACTGCACTTGAAGGAGCT	CTCAACCTCAAGACCAGTCAGT
CsWRKYIIc4	TGATGTAGAAGAAATTGGAGTTGTG	ACATACCAGTAATTGGGTTTCCA
CsWRKYIIc5	TGAGATAACGCGTAGTCCCAATAG	CTGTGGTGAATTAGTTTAGTGCAT
CsWRKYIIc6	CGTGGTGGCTACAGAGACAT	GTACTTACGTGAGACTGGTTAGCC
CsWRKYIIc7	TTCATACTTTGCTCTCTCCCTT	TGAGTTGTTCTTGTACAAAGAGGT
CsWRKYIIc8	AGAGACCTTAGACAAATCTTCCTGT	ATAAAATTAGCAATTGAAAGGGGCT
CsWRKYIIc9	TGACCTTAGAGTCAAACCTATCT	TATGAATTGTCCCATACCTGACT
qRT- CsWRKYIIc1	GAGGAAATATGGACAGAAAGCTG	GCATTCCTTCGTAAGTTGTCAC	荧光定量qRT-PCR
qRT- CsWRKYIIc2	GAATCATGCCAGAAAGTGCC	GGTGTTGGGTTGTGAAAATAGG
qRT- CsWRKYIIc3	TCCACAACCTCAATTCCAACCG	AGCAAAGACGATGACAGTGA
qRT- CsWRKYIIc4	ACCACAATCTCAACCTCAACC	CGACACAATGACAGAGCAAAAG
qRT- CsWRKYIIc5	CAGCATTGTCACCATACAGTTG	AGGCAGCTTCATGATTGACTAG
qRT- CsWRKYIIc6	AAGCCACTTGACTCCTTCAG	TGGGAAGTTGAGGATTTGGC
qRT- CsWRKYIIc7	TTCGGGTTCATGGACTTACTG	AGTCATGGGCTGGTTATTCAAG
qRT- CsWRKYIIc8	AGGTCGTTTGAAGATCCATCAG	CCACATTACCCCTCAGAGTTG
qRT- CsWRKYIIc6	TCGATCTCTTCCTCGTCTACTG	CCTCCATCTTCCACCTCTTTT
qRT- CsWRKYIIc7	GCCATCTTTGATTGGAATGG	GGTGCCACAACCTTGATCTT
qRT- CsWRKYIIc8	AGGTCGTTTGAAGATCCATCAG	CCACATTACCCCTCAGAGTTG
qRT- CsWRKYIIc9	TCGATCTCTTCCTCGTCTACTG	CCTCCATCTTCCACCTCTTTT
qRT- Csβ-actin	GCCATCTTTGATTGGAATGG	GGTGCCACAACCTTGATCTT
pGBKT7-CsWRKYIIc1	TCAGAGGAGGACCTGCATATGATGGACAACTACTCATCAAT	CGGCCTCCATGGCCATATGAAATGCCGTGTAAATTTG	构建酵母载体 Constructing yeast vectors
pGBKT7-CsWRKYIIc2	TCAGAGGAGGACCTGCATATGATGGAGAAGAAGAAACAGGA	CGGCCTCCATGGCCATATGCTCTTCTTTTGGCTCCTT
pGBKT7-CsWRKYIIc3	TCAGAGGAGGACCTGCATATGATGCTTGTTGTTGTGAGTGA	CGGCCTCCATGGCCATATGCTCGGGTTTTGCCTCCTT
pGBKT7-CsWRKYIIc4	TCAGAGGAGGACCTGCATATGATGGAGAGTAAAGAAGCTGT	CGGCCTCCATGGCCATATGGTCATCCTTTGTAACAAG
pGBKT7-CsWRKYIIc5	TCAGAGGAGGACCTGCATATGATGGATGAGAACGACAGAGT	CGGCCTCCATGGCCATATGTTGATTGCGCATTCCAGG
pGBKT7-CsWRKYIIc6	TCAGAGGAGGACCTGCATATGATGGATGATGATGATAAGGA	CGGCCTCCATGGCCATATGCTGATTGCGCATTCCAGG
pGBKT7-CsWRKYIIc7	TCAGAGGAGGACCTGCATATGATGGAGAGGAAACAAGCTGT	CGGCCTCCATGGCCATATGTTTGATGAGCCAAATTTC
pGBKT7-CsWRKYIIc8	TCAGAGGAGGACCTGCATATGATGTCTGATGAACACAGAGA	CGGCCTCCATGGCCATATGTGGCTCTTGTTTAAGAAA
pGBKT7-CsWRKYIIc9	TCAGAGGAGGACCTGCATATGATGTCTGATGAACCAGGAGG	CGGCCTCCATGGCCATATGTGGCTCTTGTTTAAAAAA

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转录因子 Transcription factor	基因ID Gene ID	开放阅读框 Open Reading Frame	氨基酸数量Amino acids number	相对分子质量 Molecular weight	理论等电点 Isoelectric point	平均疏水性Grand average of hydropathy	核定位预测值 NLSs prediction score	亚细胞定位预测Subcellular localization prediction
CsWRKYIIc1	TEA006586.1	561	186	21.098	9.28	-0.854	5.6	细胞核 Nucleus
CsWRKYIIc2	TEA028473.1	960	319	35.606	6.83	-0.872	4.6	细胞核 Nucleus
CsWRKYIIc3	TEA008513.1	936	311	34.324	6.54	-0.692	7.7	细胞核 Nucleus
CsWRKYIIc4	TEA017544.1	978	325	36.203	8.21	-0.602	4.4	细胞核 Nucleus
CsWRKYIIc5	TEA001162.1	897	298	32.070	5.79	-0.790	4.3	细胞核 Nucleus
CsWRKYIIc6	TEA022377.1	912	303	33.502	5.49	-0.894	4.3	细胞核 Nucleus
CsWRKYIIc7	TEA007197.1	720	239	27.123	8.06	-0.789	4.5	细胞核 Nucleus
CsWRKYIIc8	TEA023233.1	1008	335	36.952	6.00	-0.840	7.9	细胞核 Nucleus
CsWRKYIIc9	TEA001873.1	969	322	35.717	6.31	-0.762	5.5	细胞核 Nucleus

顺式作用元件 Cis-element	元件数量 Cis-element number									功能 Function
顺式作用元件 Cis-element	W1	W2	W3	W4	W5	W6	W7	W8	W9	功能 Function
ABRE	4	3	3	7	4	10	2	3	2	参与脱落酸反应性的顺式作用元件 Cis-acting elements involved in abscisic acid reactivity
ARE	5	2	3	1	1	2	3	2	8	厌氧诱导所必需的顺式调节元件 Cis-regulating elements necessary for anaerobic Induction
Box 4	4	5	2	1	4		7	5	4	光响应DNA模块部分 Optical response DNA module part
CGTCA-motif	1	1	1	3		4	4	5		参与MEJA反应的顺式调节元件 Cis-regulating elements involved in MEJA reaction
ERE			4	1	3	1		4	2	参与乙烯反应的顺式调节元件 Cis-regulating elements involved in ethylene reaction
G-box	3	2	1	4	2	4	1	3		光应答元件Cis-regulating elements involved in light reaction
MBS		2		1	1	2	1	1	1	干旱诱导MYB结合位点Drought-induced MYB binding site
MYB	2	6	3	1	2	1	3	2	4	参与干旱、高盐和低温诱导Drought/high salt/low temperature-induced
MYC	2	7	3		2	5	2	4	2	干旱应答元件 Cis-regulating elements involved in drought reaction
STRE		3	4	4		5		1	2	参与热响应 Heat stress responsiveness
TGACG-motif	1	1	1	3	1	4	4	5		参与MEJA反应的顺式调节元件 Cis-regulating elements involved in MEJA reaction
W box				4	2		3			防卫和胁迫应答元件 Cis-acting elements involved in defense and stress response