CAJ | 학술논문

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pOsWRKY 7-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.
WRKY 단백시식물중일류중요적전록인자,불부삼여식물생장발육적조공,환삼여식물대각충생물화비생물협박적향응.본연구종수도일본청중분리 OsWRKY7기인적편마서렬(CDS),병극륭기계동자서렬진행표체연구.수선통과실시정량 PCR 적방법검측불동조직중 OsWRKY7기인적상대표체량,결과표명,OsWRKY7재협편중적표체수평교고,차개화기적검협중표체량고우7 d 묘령적유협.진일보장 OsWRKY7계동자여 GUS 보고기인융합,구건료식물표체재체pOsWRKY 7-GUS ,병장차재체전화일본청.전기인식주불동조직염색분석결과현시,해계동자재식주적주근첨、협편、영각중유 GUS 활성,기중협편상가견전협범위분포적대량람색반점,저사염색결과여실시정량 PCR 적결과일치.진일보적접균화격소처리환현시,OsWRKY7계동자재근화협중적표체균수수도백협고병균[Xanthomonas oryzae pv．Oryzae (Xoo )]P10생리소충침염적유도,동시환수외원시가적세포분렬소화생장소유도,이수양산칙회억제기재근화협중적표체.차외,아문환장 OsWRKY7기인적 CDS 서렬분별여록색형광단백화효모 GAL4적 DNA 결합역융합,대해기인진행수도경원생질체아세포정위분석화효모자격활검험,결과현시해기인정위우세포핵중병구유전록자격활활성.상술결과표명 OsWRKY7구유명현적전록격활인자특정,기흔가능삼여료수도대백협고균적방어반응이급대다충격소신호적전도과정.
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 pOsWRKY 7-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.