水稻OsWRKY7基因的表达研究

doi:10.3969/j.issn.1001G7216.2015.06.001

中国水稻科学 ›› 2015, Vol. 29 ›› Issue (6): 559-570.DOI: 10.3969/j.issn.1001G7216.2015.06.001

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水稻OsWRKY7基因的表达研究

李茹^1,², 周洁², 李冬月^2,³, 王栩鸣², 杨勇², 余初浪², 程晔², 严成其^2,^*(), 陈剑平^1,^2,^*()

¹南京农业大学植物保护学院,南京210095
²浙江省植物有害生物防控重点实验室—省部共建国家重点实验室培育基地/农业部植物保护生物技术重点实验室/浙江省植物病毒学重点实验室/浙江省农业科学院病毒学与生物技术研究所,杭州 310021
³浙江师范大学化学与生命科学学院, 浙江金华321004

收稿日期:2015-04-22 修回日期:2015-07-18 出版日期:2015-10-25 发布日期:2015-11-10
通讯作者: 严成其,陈剑平
作者简介:
^*通讯录作者：E-mail:jpchen2001@126.com，yangchengqi@163.com
基金资助:
浙江省自然科学基金重点项目(Z14C140001)转基因生物新品种培育重大专项(2011ZX08009-003-001)

Expression of OsWRKY7 in Rice

Ru LI^1,², Jie ZHOU², Dong-yue LI^2,³, Xu-ming WANG², Yong YANG², Chu-lang YU², Ye CHENG², Cheng-qi YAN^2,^*(), Jian-ping CHEN^1,^2,^*()

¹College of Plant Protection,Nanjing Agricultural University,Nanjing 210095, China
²State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Key Laboratory of Biotechnology in Plant Protection, Ministry of Agriculture/Zhejiang Provincial Key Laboratory of Plant Virology/Institute of Virology and Biotechnology,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China
³College of Chemistry and Life Sciences,Zhejiang Normal University,Jinhua 321004,China

Received:2015-04-22 Revised:2015-07-18 Online:2015-10-25 Published:2015-11-10
Contact: Cheng-qi YAN, Jian-ping CHEN
About author:
^*Corresponding author：E-mail:jpchen2001@126.com，yangchengqi@163.com

摘要/Abstract

摘要：

WRKY蛋白是植物中一类重要的转录因子,不仅参与植物生长发育的调控,还参与植物对各种生物和非生物胁迫的响应。本研究从水稻日本晴中分离OsWRKY7基因的编码序列(CDS),并克隆其启动子序列进行表达研究。首先通过实时定量PCR的方法检测不同组织中OsWRKY7基因的相对表达量,结果表明,OsWRKY7在叶片中的表达水平较高,且开花期的剑叶中表达量高于7 d苗龄的幼叶。进一步将OsWRKY7启动子与GUS报告基因融合,构建了植物表达载体pOsWRKY7-GUS,并将此载体转化日本晴。转基因植株不同组织染色分析结果显示,该启动子在植株的主根尖、叶片、颖壳中有GUS活性,其中叶片上可见全叶范围分布的大量蓝色斑点,这些染色结果与实时定量PCR的结果一致。进一步的接菌和激素处理还显示,OsWRKY7启动子在根和叶中的表达均受水稻白叶枯病菌[Xanthomonas oryzae pv. Oryzae(Xoo)]P10生理小种侵染的诱导,同时还受外源施加的细胞分裂素和生长素诱导,而水杨酸则会抑制其在根和叶中的表达。此外,我们还将OsWRKY7基因的CDS序列分别与绿色荧光蛋白和酵母GAL4的DNA结合域融合,对该基因进行水稻茎原生质体亚细胞定位分析和酵母自激活检验,结果显示该基因定位于细胞核中并具有转录自激活活性。上述结果表明OsWRKY7具有明显的转录激活因子特征,其很可能参与了水稻对白叶枯菌的防御反应以及对多种激素信号的转导过程。

关键词: OsWRKY7基因, 启动子, 表达特性, 亚细胞定位, 转录活性

Abstract:

WRKY proteins are important transcription factors in plants. They are involved in various plant developmental processes, as well as in coping with diverse biotic and abiotic stresses. In this study,we cloned the coding sequence (CDS) and upstream promoter of OsWRKY7 from Nipponbare to analyze its expression patterns. We first analyzed the relative expression level of OsWRKY7 in different tissues by quantitative real-time PCR(qRT-PCR), and the result showed that OsWRKY7 is mainly expressed in leaves, with a higher expression level in flag leaves than that in seedling leaves. We then constructed the pOsWRKY7-GUS expression vector by fusing the putative promoter with GUS reporter gene and transformed the vector into Nipponbare. Subsequent GUS staining showed that OsWRKY7 promoter had activity in primary root tip, leaf blade and glume. Consistent to the qRT-PCR result, massive GUS spots were stained on the entire leaf blade. We also characterized the inducibility of the pOsWRKY7-GUS transgenic plants to pathogen infection and hormone treatment. The results showed that the GUS activities in both leaf and root are up-regulated after inoculation with rice bacterial blight pathogen [Xanthomonas oryzae pv. Oryzae (Xoo)] strain P10, as well as after exogenous application of cytokinin and auxin, while salicylic acid treatment represses GUS activity in both leaf and root. Finally, we fused the CDS of OsWRKY7 with the green fluorescent protein and the GAL4 DNA binding domain, respectively to analyze its subcellular localization in rice and transcriptional activity in yeast. The results showed that OsWRKY7 was localized exclusively to the nucleus of rice stem protoplasts, and has transcriptional self-activation activity in yeast. All these data suggested that OsWRKY7 might act as a transcriptional activator in bacterial blight defense and diverse hormone signal transduction pathways.

Key words: OsWRKY7, promoter, expression characteristic, sublocalization, transcriptional activity

中图分类号:

Q786

李茹, 周洁, 李冬月, 王栩鸣, 杨勇, 余初浪, 程晔, 严成其, 陈剑平. 水稻OsWRKY7基因的表达研究[J]. 中国水稻科学, 2015, 29(6): 559-570.

Ru LI, Jie ZHOU, Dong-yue LI, Xu-ming WANG, Yong YANG, Chu-lang YU, Ye CHENG, Cheng-qi YAN, Jian-ping CHEN. Expression of OsWRKY7 in Rice[J]. Chinese Journal OF Rice Science, 2015, 29(6): 559-570.

图/表 7

表1 本研究使用的引物序列及UPL探针

Table 1 The primers sequences and UPL probes used in this study.

引物 Primer	引物序列 Primer sequence	UPL探针 UPL probe
OsWRKY7-RT-F	TGATCGAGATAGCAGCCTCTT	105
OsWRKY7-RT-R	CCAGCTACATCCAAAATGACC
OsActin-RT-F	CAACACCCCTGCTATGTACG	158
OsActin-RT-R	CATCACCAGAGTCCAACACAA
OsWRKY7-Pr-F	CGACGCCCTATCCGATTCCAA
OsWRKY7-Pr-R	GCGCGAAAGCTCCGTATTTATAAG
OsWRKY7-Pr-FW	CCGGGGATCCTCTAGCGACGCCCTATCCGATTC
OsWRKY7-Pr-RV	TACCGAATTCGTCGAGCGCGAAAGCTCCGTATT
OsWRKY-CDS-F	TATGGCGGCAGTCGGCGCG
OsWRKY-CDS-R1	TTTAATTGAGAGAACCTGGCGGCTGCGTG
OsWRKY-CDS-R2	TATTGAGAGAACCTGGCGGCTGCGTG
OsWRKY-CDS-FW	AGGAGGACCTGCATATGGCGGCAGTCGGCGCG
OsWRKY-CDS-RV	GCAGGTCGACGGATCTTAATTGAGAGAACCTGGCG

图1 OsWRKY7基因编码框和启动子序列的克隆和载体构建

Fig. 1. Cloning of OsWRKY7 coding and promoter sequences for vector construction.

图2 OsWRKY7的组织表达分析

Fig. 2. Tissue expression of OsWRKY7.

图3 白叶枯菌P10生理小种处理对OsWRKY7启动子的表达调控

Fig. 3. Response of the OsWRKY7 pormoter to innoculation of Xoo P10 race.

图4 植物激素激动素(KT)、乙酰水杨酸(SA)和3-吲哚乙酸(IAA)处理对OsWRKY7启动子的表达调控

Fig. 4. Responses of the OsWRKY7 pormoter to phytohormones treatment of kinetin(KT), salicylic acid(SA) and 3-indole-acetic acid(IAA).

图5 OsWRKY7的亚细胞定位

Fig. 5. Subcellular localization of OsWRKY7.

图6 OsWRKY7的转录激活活性验证

Fig. 6. Transcriptional activation activity of OsWRKY7.

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水稻OsWRKY7基因的表达研究

Expression of OsWRKY7 in Rice

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