脱腺苷酸酶基因CpCAF1对蜡梅花发育和非生物胁迫响应的调控

The proper regulation of gene expression is crucial for essentially all biological processes, including plant growth and development,as well as its response to environmental stresses. Although gene expression could be regulated at different processes, controlled RNA degradation is the essential one that allows rapid change in a cell's gene expression profile. CCR4-associat-ed factor 1 (CAF1), a subunit of the CCR4-NOT complex, is one of the most important deadenylases that has been found to be present and active in the process of mRNA decay. It plays an important role in the regulation of gene expression and has great effect on biological characteristics. A putative Chimonanthus praecox homolog of the CAF1 gene, CpCAF1, with a full length of 1KB, has been acquired in our previous study. This gene is predominantly expressed in floral organs in Chimonanthus praecox. Over-expression of CpCAF1 results in faster growth and a delay of floral development in tobacco. In the project, we hope to explore the function of CpCAF1 and its complex regulatory network during Chimonanthus praecox development and response to abiotic stresses. Experiments of CpCAF1 protein structural analysis, gene silencing, subtractive cDNA library construction analysis, hormone induced expression, environmental stress responses, promoter regulatory elements and activity analysis will be performed in the study. The structural basis of CpCAF1's function and functional differences, substrate specificity of CpCAF1 mediating mRNA degradation, possible biological characters affected by CpCAF1, and regulation of CpCAF1 expression will be demonstrated. With the results of above experiments, the regulatory network of Chimonanthus praecox CpCAF1 will be further elucidated. The research is significantly important to explore the unique mechanisms of floral development and stress resistance in Chimonanthus praecox and other winter flowering plants. It will also provide a theoretical basis for developing technology of regulating Chimonanthus praecox blooming.

mRNA降解是基因表达调控的关键步骤， CAF1(CCR4-associated factor 1) 是参与mRNA降解的主要脱腺苷酸酶，在调控基因表达和影响生物学性状方面起重要作用。申请人从蜡梅中获得了脱腺苷酸酶基因CpCAF1，该基因在蜡梅花器官中优势表达，烟草中过表达表现出明显的生长加快和花发育延迟。为探明CpCAF1的基因功能及其调控网络，拟通过蛋白活性与结构解析、基因沉默、差减文库构建分析、激素等胁迫响应、启动区调控元件与活性分析等实验，明确蜡梅CpCAF1存在的功能活性以及功能差异的结构基础、CpCAF1基因调控的主要基因和生物学性状、CpCAF1基因表达与调控特点，进而初步探明蜡梅中CpCAF1基因的调控通路。项目完成有望为揭示蜡梅等冬季开花植物的花发育和抗逆性形成机制的独特性提供重要线索，为开发蜡梅开花调控技术提供理论依据，为揭示植物CAF1基因调控机制提供佐证。

为了探索mRNA降解在蜡梅冬季开花过程中的调控作用，本研究克隆了蜡梅脱腺苷酸酶CpCAF1基因cDNA全长，编码蛋白预测分子式为C1371H2087N349O410S11，分子量为30.37KD，等电点pI是4.70。该基因的编码蛋白为CCR4-NOT transcription complex subunit 7，属于DEDD核酸酶家族的DEDDh亚族。其活性区由3个天门冬甘酸（D）、1个谷氨酸（E）以及邻近的组氨酸（h）组成。构建了蜡梅CpCAF1基因的原核表达载体PET32a(+)-CpCAF1，并转入表达菌株BL21(DE 3)。进一步开展PAT（poly(A) test）试验，证明重组蛋白具有体外脱腺苷酸酶活性。将构建好的植物表达载体pCAMBIA2301G-CpCAF1转入农杆EHA105中，利用花序侵染法转化拟南芥，获得T4代转基因拟南芥株系。通过表型观察、低温胁迫表现及生理指标检测表明，CpCAF1基因可能参与开花物候和抗寒性形成的调控，超表达植株开花提早和抗寒性提高。利用Clontech公司提供的酵母双杂交文库构建试剂盒构建了基于蜡梅花cDNA酵母文库。库容为4.38 x106cfu，滴度为3.9 x107cfu/ml，200-2500bp的片段占所有片段的94.9%，500bp以上片段占所有片段的59%。在此基础上筛选到CpCAF1可能作用的靶蛋白编码基因。时空表达特征分析表明，CpCAF1在花器官中表达量相对较高，低温响应迅速，随着花器官的不断发育，表达量逐渐升高，其靶蛋白的表达与CpCAF1有一定的关联性。利用genomic walking技术克隆到1909 bp的CpCAF1启动子CpCAF1 pro，瞬时表达证实CpCAF1启动子CpCAF1 pro具有启动活性，通过PLANTCARE分析表明启动子区有大量环境胁迫和激素信号响应元件，说明CpCAF1的表达可能受多种激素和胁迫信号的调控。项目结果有利于进一步了解植物CAF1基因功能，同时为蜡梅冬季开花过程中花朵发育与抗寒性关联提供一定的实验依据，从而加深对蜡梅冬季开花独特性的认识。