*通讯录作者：E-mail:jpchen2001@126.com，yangchengqi@163.com
收稿日期: 2015-04-22
修回日期: 2015-07-18
网络出版日期: 2015-11-10
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
*Corresponding author：E-mail:jpchen2001@126.com，yangchengqi@163.com
Received date: 2015-04-22
Revised date: 2015-07-18
Online published: 2015-11-10
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。
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 . DOI: 10.3969/j.issn.1001G7216.2015.06.001
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.
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