Subcellular Localization Analysis of Rice Protein Kinase Gene OsCIPK5 and Generation of Its RNAi Transgenic Plants
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摘要: 在植物生长发育中，CIPKs（CBL-interacting protein kinases）在胁迫信号转导和增强抗逆途径中发挥着重要作用，而OsCIPK5的具体功能还未知。为了研究OsCIPK5的功能，本研究从日本晴水稻中成功克隆了OsCIPK5基因。用生物信息学方法对其编码的蛋白进行分析，结果表明OsCIPK5含有2个功能区即激酶活性区和NAF区，OsCIPK5与5种植物的CIPK5同源性较高，而与短花药野生稻CIPK5的同源性最高（94%），亲缘关系最近。利用植物生理学方法对水稻进行低钾处理，结果表明水稻根中OsCIPK5受低钾诱导表达，而叶片中OsCIPK5表达量没有变化；同时构建了OsCIPK5与黄色荧光蛋白基因融合的植物瞬时表达载体，共聚焦显微镜观察显示，OsCIPK5编码的蛋白主要定位在细胞核、细胞膜，还以颗粒状结构不规则分布在细胞质中。进一步构建了OsCIPK5的RNAi载体，通过水稻遗传转化体系获得26株阳性转基因水稻。荧光定量PCR（Real-time qPCR）分析表明，T1代转基因水稻中OsCIPK5的表达量与野生型相比显著降低。OsCIPK5的生物信息学分析、植物生理学分析、亚细胞定位以及RNAi转基因水稻的获得为研究OsCIPK5的功能奠定了基础。
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关键词:
- 蛋白激酶OsCIPK5 /
- 生物信息学分析 /
- 表达模式 /
- 亚细胞定位 /
- RNAi
Abstract: Plant CBL-interacting protein kinases (CIPKs) play an important role in stress signaling transduction and enhancing plant stress tolerance. However, the functions of OsCIPK5 in the rice (Oryza sativa) have not been studied. In order to study the functions of OsCIPK5, RT-PCR method was used to clone OsCIPK5 gene from rice leaf (Oryza sativa Japonica Group cultivar Nipponbare).The bioinformatics analysis showed that OsCIPK5 contained two functional domains, N-terminal kinase activation loop domain and C-terminal NAF domain, which were the same as other CIPK that had been reported.OsCIPK5 was highly homologous in amino acid with other five plant species, especially with Oryza brachyantha, which was about 94%. By Real-time PCR approach the expression pattern of OsCIPK5 was detected in the rice seedlings responses to potassium. The result showed that the expression of OsCIPK5 in root was up-regulated under low potassium treatment, but not in leaf. The recombinant expression plasmid pEarleyGate101-OsCIPK5 contained yellow fluorescent protein (YFP) was transformed into GV3101 and then the positive clones were infiltrated into the epidermal leaves of Nicotiana benthamiana. The results revealed that the OsCIPK5 protein localized on the nucleus, cytolemma and cytoplasm. The RNAi vector containing OsCIPK5 specific gene was constructed and transferred into EHA105 (Agrobacterium tumefaciens), then rice transformation was conducted through agrobacterium-mediated system. T0 RNAi transgenic seedlings of OsCIPK5 were obtained and 26 plants were identified to be positive.The expression of OsCIPK5 in transgenic plants was obviously reduced compared with that in wild type by Real-time PCR analysis.These results including bioinformatics analysis, expression pattern, subcellular localization analysis and transgenic rice may be useful in study on the important role of OsCIPK5 in the process of plant growth.-
Key words:
- OsCIPK5 /
- bioinformatic analysis /
- expression pattern /
- subcellular localization /
- RNAi
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图 1 OsCIPK5基因的RT-PCR扩增结果
注：M为DNA分子标记Trans2K Plus；1为OsCIPK5 RT-PCR扩增产物。
Figure 1. RT-PCR result of OsCIPK5
图 2 OsCIPK5的氨基酸序列分析
注：A为6种植物CIPK5氨基酸序列的多重性比对；B为6种植物CIPK5的氨基酸同源性分析；C为6种植物CIPK5的系统发育分析。
Figure 2. Analysis of the amino acid sequence of OsCIPK5
图 3 水稻叶片和根中OsCIPK5低钾处理表达分析
Figure 3. Real-time PCR analysis of OsCIPK5 expression level in leaf and rot of rice under low K treatment
图 4 OsCIPK5蛋白在本氏烟下表皮细胞中的定位
注：A为黄光荧光；B为叶绿体；C为融合。Bar=8 μm。
Figure 4. Localization in Nicotiana benthamiana cell of OsCIPK5
图 5 OsCIPK5 RNAi转基因水稻的PCR检测
注：M为DNA分子标记MakerⅢ；1~9为OsCIPK5 RNAi转基因水稻PCR检测；CK为阴性对照。
Figure 5. PCR detection of OsCIPK5 RNAi in transgenic rice plants
图 6 qPCR检测转基因水稻中OsCIPK5相对表达量
注：WT为野生型水稻。
Figure 6. OsCIPK5 gene relative expression level in transgenic rice by qPCR
表 1 PCR引物
Table 1. PCR primers
引物名称 引物序列(5′-3′) 引物用途 CIPK5-F ATG GAG AAG AAG GCG TCC ATC OsCIPK5的扩增 CIPK5-R TTA AAT GGC ATG TCT CGA GAT TG OsCIPK5 gene cloning G418-F GTC CCG CTC AGA AGA ACT CGT C 转基因水稻的检测 G418-R CGC TGA AAT CAC CAG TCT CTC TC Detection of transgenic rice q-CIPK5-F ATA GGG GCT GTG GAC TTC TGT OsCIPK5表达量检测 q-CIPK5-R GAC ACT TTG AGG TTG CCA TTC Detection of relative expression level of OsCIPK5 GAPDH-F AAG CCA GCA TCC TAT GAT CAG ATT 内参基因检测 GAPDH-R CGT AAC CCA GAA TAC CCT TGA GTT T Detection of internal reference gene GAPDH 
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