【作者】 李冰；

【导师】 周人纲； 孙大业；

【作者基本信息】 河北师范大学 ， 细胞生物学， 2003， 博士

【摘要】 先前的工作已经表明Ca²⁺-CaM信号系统影响热激基因的表达。本文实验主要以玉米为材料，在热激转录因子层次进一步研究Ca²⁺及CaM如何在热激信号转导中调节热激基因表达。 本课题以[г-³²P]-ATP标记的HSE为探针，通过凝胶阻滞分析研究Ca²⁺和CaM与HSF的DNA结合活性的关系。在体外44℃热激玉米幼苗全细胞提取液可诱导HSF的DNA结合活性。热激诱导的HSF的DNA结合活性具有时间依赖性，44℃热激30min时HSF的活性最高。竞争结合实验表明HSF与合成的HSE的结合具有专一性。44℃热激条件下以Ca²⁺螯合剂EGTA处理玉米幼苗全细胞提取液可抑制HSF的DNA结合活性，而回加CaCl₂又可恢复HSF的活性；在非热激条件下直接以CaCl₂处理则可代替热激激活HSF的活性，其中1mM CaCl₂的作用最明显，而相同浓度的MgCl₂无此作用。CaM拮抗剂W7和CPZ都可抑制热激诱导的HSF的DNA结合活性，而W7的类似物W5无此抑制作用。兔抗CaM血清也明显抑制44℃热激诱导的HSF的DNA结合活性，而回加CaM又可恢复其活性；热激条件下直接以CaM处理可促进HSF的DNA结合活性，同样浓度的S-100和BSA无明显作用；另外，在非热激条件下外加CaM还可替代热激诱导HSF的DNA结合活性，而同样浓度的BSA处理无此作用。以上结果在植物中进一步证实了Ca²⁺可促进HSF的DNA结合活性，并首次表明CaM也可促进HSF的DNA结合活性，由此提出Ca²⁺-CaM信号系统影响热激基因的表达可能是通过调节HSF的活性而实现的。 前人的研究表明，HSP70在热激基因表达中起负调节剂作用，在非热激条件下，它通过与HSF结合成复合体抑制HSF的活性。本文实验用免疫共沉淀法在细胞提取液中检测到了HSP70与HSF复合体的存在，并通过凝胶阻滞分析法研究了HSP70对HSF的DNA结合活性的影响，在植物中为HSP70的负调节作用提供了支持证据。 先前我们在玉米中，通过体外结合和竞争抑制实验证明了热激蛋白家族中最重要的成员之一纯化的细胞质HSP70能与CaM结合，并且证明了玉米细胞质HSP70中存在一个非常保守的21个氨基酸组成的CaM结合序列。这种高度的保守性意味着CaM与HSP70结合可能具有重要的生物学功能。本文实验通过免疫共沉淀法在细胞提取液中检测到了HSP70与CaM复合博士学位论文物的存在，并表明热激后的细胞提取液中HSP7O与CaM复合物的含量有所增加。说明CaM影响热激基因表达可能是通过与HSP70结合起作用。更多还原

【Abstract】 Our previous work has shown that Ca2+-CaM signal system is involved in HS gene expression. Herein, we mainly focus on the mechanism by which Ca2+-CaM plays a role at the level of HSF. Maize was used as a main experimental material.Gel mobility shift assay was employed to study the relationship between Ca2+-CaM and DNA-binding activity of heat shock transcription factor (HSF). The binding activity of the HSF to heat shock element (HSE) was induced by heat shock (44 ℃) of a maize whole cell extract. DNA-binding activity of the HSF increased in a time-dependent manner, it increased gradually with time of HS and reached its maximum after 30 min of HS. Competition binding experiment indicates that the binding of the HSF to synthetic HSE is specific. Under HS at 44℃, addition of the calcium ion chelator EGTA to a whole cell extract reduced the binding of the HSF to HSE. Re-addition of CaCl2 to the sample pretreated by EGTA restored the binding activity of the HSF to HSE obviously. DNA-binding activity of the HSF was also induced by directly adding Ca2+ to a whole cell extract at non-heat-shock temperature, but not by the same concentration of MgCl2. Effect of 1mM CaCl2 is most prominent. During HS at 44 ℃, the CaM antagonists W7 and CPZ also inhibited DNA-binding activity of the HSF in a concentration-dependent manner, but not by W5, an inactive structural analogue of W7. Addition of antiserum specific to CaM to a whole cell extract reduced binding of the HSF to HSE at 44℃. Re-addition of CaM to the cell extract pretreated with antiserum might restore the ability of the HSF to bind to HSE. DNA-binding activity of the HSF was promoted by directly adding CaM to a whole cell extract at 44 ℃, but not by the same concentration of S100 and BSA. Moreover, at non-heat-shock temperature, DNA-binding activity of the HSF was also induced by directly adding CaM to a whole cell extract but not by the same concentration of BSA. Our observations further confirm the role of Ca2+ in activation of HSF in plant and provide the first example of the role of CaM in regulation of the DNA-binding activity of the HSF. These results suggest thatCa2+ and CaM were involved in HS genes expression likely thlough regulating the activity of HSRSome studies have suggested that HSP70 is a negative regulatory factor. It represses activity of HSF by formation of complex with HSF under non-heat-shock condition. The result of co-immunoprecipetation showed that HSF-HSP70 complex existed in cytoplasmic extract. Gel mobility shift assay was used to study the affect of HSP70 on the activity of HSF. These results provide evidence for negative regulation role of HSP70 in plant.Our previous work has also shown that there is a CaM-binding site within maize cytoplasmic HSP70 and that HSP70 binds CaM in a Ca2+-dependent manner (Sun et al, 2000). The high conservation of CaM-binding suquence implies that the binding of CaM to HSP70 might have an important biological function. It was shown that CaM-HSP70 complex existed in heat shocked cytoplasmic extract by co-immunoprecipetation experiment. The result indicates that CaM regulates HS gene expression likely through binding to HSP70.更多还原