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刚地弓形虫RH株ROP11基因的克隆与真核表达载体的构建 被引量:7
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作者 张晓磊 张进顺 +5 位作者 朱晓波 王春苗 贾晓晖 贾天军 徐云鹏 高雪 《中国病原生物学杂志》 CSCD 北大核心 2013年第11期1002-1004,1016,共4页
目的构建刚地弓形虫(Toxoplasma gondii)棒状体蛋白11(ROP11)的真核表达重组质粒并在真核细胞中表达目的蛋白。方法设计合成弓形虫ROP11基因引物,运用RT-PCR方法扩增ROP11基因并克隆至真核表达质粒pcDNA3.1(+),构建重组表达质粒pcDNA3.1... 目的构建刚地弓形虫(Toxoplasma gondii)棒状体蛋白11(ROP11)的真核表达重组质粒并在真核细胞中表达目的蛋白。方法设计合成弓形虫ROP11基因引物,运用RT-PCR方法扩增ROP11基因并克隆至真核表达质粒pcDNA3.1(+),构建重组表达质粒pcDNA3.1-ROP11。将重组质粒转染HeLa细胞,采用间接免疫荧光法检测目的蛋白表达。结果 RT-PCR扩增弓形虫ROP11基因片段为1 548bp,与预期大小相符。构建的重组质粒pcDNA3.1-ROP11经PCR及EcoRⅠ和NotⅠ双酶切鉴定正确。重组质粒测序后与GenBank报道的ROP11基因比对,核苷酸序列同源性和推导氨基酸序列同源性均为99%。免疫荧光检测显示,在重组质粒转染的HeLa细胞胞浆观察到黄绿色荧光,对照组无荧光。结论成功构建了真核表达质粒pcDNA3.1-ROP11,该重组质粒能够在真核细胞中表达目的蛋白,为弓形虫核酸疫苗的研制奠定了基础。 展开更多
关键词 刚地弓形虫 rop11 真核表达质粒
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ROP11 GTPase is a Negative Regulator of Multiple ABA Responses in Arabidopsis 被引量:16
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作者 Zixing Li Jun Kang Ning Sui Dong Liu 《Journal of Integrative Plant Biology》 SCIE CAS CSCD 2012年第3期169-179,共11页
The phytohormone abscisic acid (ABA) plays crucial roles in plant development and plant responses to environmental stresses. Although ABA receptors and a minimal set of core molecular components have recently been d... The phytohormone abscisic acid (ABA) plays crucial roles in plant development and plant responses to environmental stresses. Although ABA receptors and a minimal set of core molecular components have recently been discovered, understanding of the ABA signaling pathway is still far from complete. In this work, we characterized the function of ROP11, a member of the plant-specific ROP small GTPases family, in the ABA signaling process. ROP11 is preferentially expressed in guard cells in all plant organs with stomata. Expression of a constitutively active ROP11 (CA-ROP11) suppresses ABA-mediated responses, whereas reduced expression of ROP11 or expression of its dominant-negative form (DN-ROP11) causes the opposite phenotypes. The affected ABA-mediated responses by ROP11 include seed germination, seedling growth, stomatal closure, induction of ABA-responsive genes, as well as plant response to drought stress. Furthermore, we showed that ROP11 and its closest-related family member, ROP10, act in parallel in mediating these responses. ABA treatment does not affect ROP11 transcription and protein abundance; however, it causes the accumulation of CA-ROP11 in the nucleus. These results demonstrated that ROP11 is a negative regulator of multiple ABA responses in Arabidopsis. 展开更多
关键词 rop11 GTPase ABA responses negative regulator rop10rop1 double mutant Arabidopsis.
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ROP11 GTPase Negatively Regulates ABA Signaling by Protecting ABI1 Phosphatase Activity from Inhibition by the ABA Receptor RCAR1/PYL9 in Arabidopsis 被引量:15
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作者 Zixing Li Zheng Li +6 位作者 Xiang Gao Viswanathan ChinnusamyI Ray Bressant Zhi-Xin Wang Jian-Kang Zhu Jia-Wei Wu Dong Liu 《Journal of Integrative Plant Biology》 SCIE CAS CSCD 2012年第3期180-188,共9页
The phytohormone abscisic acid (ABA) regulates many key processes in plants, such as seed germina- tion, seedling growth, and abiotic stress tolerance. In recent years, a minimal set of core components of a major AB... The phytohormone abscisic acid (ABA) regulates many key processes in plants, such as seed germina- tion, seedling growth, and abiotic stress tolerance. In recent years, a minimal set of core components of a major ABA signaling pathway has been discovered. These components include a RCAR/PYR/PYL family of ABA receptors, a group of PP2C phosphatases, and three SnRK2 kinases. However, how the interactions between the receptors and their targets are regulated by other proteins remains largely unknown. In a companion paper published in this issue, we showed that ROP11, a member of the plant- specific Rho-like small GTPase family, negatively regulates multiple ABA responses in Arabidopsis. The current work demonstrated that the constitutively active ROP11 (CA-ROP11) can modulate the RCAR1/PYL9-mediated ABA signaling pathway based on reconstitution assays in Arabidopsis thaliana protoplasts. Furthermore, using luciferase complementation imaging, yeast two-hybrid assays, co- immunoprecipitation assays in Nicotiana benthamiana and bimolecular fluorescence complementation assays, we demonstrated that CA-ROP11 directly interacts with ABI1, a signaling component downstream of RCAR1/PYL9. Finally, we provided biochemical evidence that CA-ROP11 protects ABI1 phosphatase activity from inhibition by RCAR1/PYL9 and thus negatively regulates ABA signaling in plant cells. A model of how ROP11 acts to negatively regulate ABA signaling is presented. 展开更多
关键词 rop11 GTPase ABA signaling RCAR1/PYL9A ABI1 Arabidopsis.
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