【作者】 张成伟；

【导师】 黎裕；

【作者基本信息】 中国农业科学院 ， 作物种质资源学， 2009， 博士

【摘要】 钙调磷酸酶B类蛋白(calcineurin B-like proteins, CBLs)是近年来鉴定出的一类植物中特有的Ca2+传感器,与其互作的蛋白CIPK(CBL-interacting protein kinase)一起在植物特定的生长发育和应答胁迫过程中起重要作用。对模式植物拟南芥和水稻的研究表明,CIPK广泛参与植物对高盐、低钾和干旱等非生物胁迫的应答反应。目前对于CIPK的研究主要集中在拟南芥和水稻上,其他植物上的研究相对较少。玉米作为世界上重要的粮食作物,干旱与土壤贫瘠等非生物胁迫是限制玉米生产的重要因素,挖掘玉米CIPK基因并进行功能研究,不但具有重要的理论意义,而且具有广泛的应用价值,可以为玉米抗逆转基因育种提供潜在的有效候选基因。本研究从玉米中克隆到一个新的CIPK基因ZmCIPK31,并对其生化属性、表达特性及功能等进行了分析。主要研究工作与结果如下:1、ZmCIPK31基因的克隆与序列分析利用RACE的方法,克隆得到ZmCIPK31全长cDNA,同时利用PCR方法得到了ZmCIPK31的基因组DNA序列和启动子序列。ZmCIPK31基因组含有14个外显子和13个内含子,蛋白结构上具有CIPK所共有的N端激酶结构域和C端NAF保守结构域。对ZmCIPK31启动子序列内的顺式作用元件进行预测分析,发现该基因启动子区域具有光应答、胁迫应答、发育相关、激素相关及其它功能未知的调控结构域。2、ZmCIPK31蛋白激酶活性分析构建原核表达重组载体pET-ZmCIPK31和pMAL-ZmCIPK31,通过原核表达分析,在大肠杆菌中成功的诱导表达了预期大小的目的蛋白。通过蛋白可溶性分析,发现His-tag-ZmCIPK31融合蛋白是不可溶性的,而MBP-ZmCIPK31融合蛋白是可溶性的。因此,将MBP-ZmCIPK31融合蛋白纯化并浓缩,用于蛋白激酶活性分析试验。蛋白自磷酸化试验表明ZmCIPK31具有激酶活性,且微量的Mn2+是有效的辅助因子。3、ZmCIPK31基因在玉米中的表达分析ZmCIPK31在正常生长条件的玉米的茎、根、幼胚、花丝、种子及叶中均有一定量的基础表达,且在花丝中的相对表达量最大。ZmCIPK31受盐、冷和PEG的诱导表达,而不受ABA和脱水处理诱导表达。4、ZmCIPK31亚细胞定位及与ZmCBLs的互作分析洋葱表皮及拟南芥原生质体中ZmCIPK31的亚细胞定位分析表明ZmCIPK31定位于细胞膜、细胞质和细胞核上。酵母双杂实验对ZmCIPK31s与ZmCBLs的互作分析及BiFC实验表明,ZmCIPK31与ZmCBL3和ZmCBL4互作,且NAF结构域是它们互作所必须的结构域;缺失了ZmCIPK31紧邻NAF结构域的C端区域的ZmCIPK31-337C除了与ZmCBL3和ZmCBL4互作外,还与ZmCBL2-1、2-2、5、6互作。5、ZmCIPK31基因转化野生型拟南芥在正常培养条件下的MS培养基、添加100mM NaCl的MS培养基以及冷处理条件下的MS培养基上,过表达ZmCIPK31的转基因拟南芥株系与野生型的种子萌发率、根长对比均没有显著差异,而在125mM和150mM NaCl的MS培养基上,转基因株系的种子萌发率显著高于野生型对照,根长与野生型没有显著差异。更多还原

【Abstract】 The calcineurin B-like proteins (CBLs) are recently identified to be a kind of plant specific calcium sensors. CBL interacts with CIPK (CBL-interacting protein kinase). CBL and CIPK were proved to play important roles in responding to stresses and regulating certain developmental processes in plants. Based on studies on model plants Arabidopsis and rice, CIPKs perticipate in responsiveness of plants to abiotic stresses, such as high salinity, low potassium and drought. However, although researches on CIPKs have made much progress in Arabidopsis and rice in the recent years, only a few studies have been made and thus related information is limited in other plant species. Maize is one of the most important cereal crops in the world, its production is limited under abitotic stresses like drought and poor soil. Cloning CIPKs in maize not only shows theoretical meanings, but also benefits to practical application, providing potential candidate genes for improving stresses torlerance by genetic transformation in maize. Here, we isolated ZmCIPK31, a new CIPK in maize. Its biochemical property, expression profiles and fuctions have been analized. The main results were as follows.1. Cloning and sequence analysis of ZmCIPK31 geneThe full length of ZmCIPK31 cDNA was obtained by using RACE method, while the genomic DNA of ZmCIPK31 with the promoter region, 2189bp upstream of ATG initiation codon, was obtained by PCR method. The genomic DNA was composed of 14 exons and 13 introns in its coding region. And it contained a serine/threonine kinase domain in N-terminal region and a NAF domain in C-terminal region. A promoter motif search of this region showed that there were motifs related to light, stress, development, auxin and others.2. Protein kinase activity of ZmCIPK31The recombinant plasmids pET-ZmCIPK31 and pMAL-ZmCIPK31 were constructed for prokaryotic expression analysis. The results showed that the predicted target protein could be induced in the recombinant plasmid transformed E. coli BL21 strain cells. Protein soluble analysis proved the fusion protein His-tag-ZmCIPK31 was not soluble, while MBP-ZmCIPK31 was soluble. Thus, the purified and concentrated soluble MBP-ZmCIPK31 fusion protein was obtained for protein kinase assay. The prtein autophosphorylation assay showed that ZmCIPK31 possessed protein kinase properties and millimolar range of Mn2+ was effective cofactor.3. Expression analysis of ZmCIPK31 gene in maizeCertain ZmCIPK31 expression level in stem, root, embryo, filament, seed and leaf of maize were detected under normal growth conditions. And the relative expression level is the highest in filament. ZmCIPK31 was regulated by NaCl, cold and PEG, but not by ABA and dehydration.4. Subcellular localization of ZmCIPK31 and interation with ZmCBLsSubcellular localization analysis in onion epidermal cells and Arabidopsis mesophyll protoplasts showed that ZmCIPK31 expressed transiently in plasma membrane, cytoplasm and the nucleus. Yeast hybrid assay and BiFC assay showed that ZmCIPK31 interacted specifically with ZmCBL3 and ZmCBL4, and the NAF domain was necessary. ZmCIPK31-337C, however, exhibited more extensive affinities with ZmCBL2-1, 2-2, 5, 6 other than ZmCBL3 and ZmCBL4.5. Over-expression of ZmCIPK31 in ArabidopsisIn MS medium with or without 4°C treatment and in MS medium with 100 mM NaCl, the seeds germination rate and root length of all five transgenic lines were just like that of the control wild-type. However, in the medium with 125 mM or 150 mM NaCl, all transgenic lines showed significantly higher germination rates than the wild-type Arabidopsis. On the contrary, there was no significant difference of the root length between transgenic lines and wild type.更多还原