【作者】 张和臣；

【导师】 尹伟伦； 夏新莉；

【作者基本信息】 北京林业大学 ， 森林培育， 2010， 博士

【摘要】 钙离子作为植物细胞中的第二信使在调节和响应外来非生物胁迫的信号转导中起着重要作用,钙信使产生后通过与钙离子结合蛋白相互作用,从而将信号继续向下游传递。在植物细胞中发现有钙调素、CDPK.CBL等多种钙离子结合蛋白。CBL蛋白作为近年来刚发现的一类钙结合蛋白,它可以通过与一类丝氨酸／苏氨酸蛋白激酶-CIPK相互作用而介导钙信号的转导。在拟南芥中对钙信号网络已经进行了深入研究,但是在其它植物特别是在林木植物研究上还未见报道。杨树基因组(毛果杨：Populus trichocarpa)测序的完成,为我们研究CBL-CIPK信号途径提供了良好平台(model).本研究利用毛果杨基因组数据库对胡杨(Populus euphratica)(一种沙漠建群树种)中的CBL-CIPK信号途径进行了系统研究。1.首先利用生物信息学手段,在毛果杨基因组中(毛果杨)鉴定和分离了10个编码CBL蛋白的基因、25个编码CIPK蛋白的基因。通过比较基因组学对毛果杨、拟南芥、水稻CBL和CIPK家族结构、结构域和进化关系做了分析,研究表明在进化过程中这两个家族在成员数量和家族结构上比较保守。但是与拟南芥相比,在毛果杨基因组中有更多的并系同源基因对(paralogous gene pairs),研究发现毛果杨多数同源基因对是通过染色体间的复制和交换产生的。毛果杨染色体间的复制和相互交换事件在该家族基因成员数量扩增过程中起了重要作用。为了进一步研究这两个家族在逆境条件下所起的作用,我们在胡杨中克隆出了10个CBL(PeCBL1-10)基因家族成员,14个CIPK(PeCIPK3α、5、6b、7、9α、11α、12b、14、1 5、16、23α.23b.24a、24b)基因家族成员,并在逆境条件下对10个PeCBL基因成员的表达作了分析。研究发现,PeCBLl,2,3,4,5,9和PeCBL10等7个成员在对外来逆境做出的调控反应中起着重要作用。研究10个CBL基因编码的氨基酸序列发现,它们除了在N端含有一段不太保守的序列之外,在C端都含有4个与钙离子结合的EF手型结构。而N端不保守的序列可能与该蛋白在细胞中的亚细胞定位有关。比较14个PeCIPK的氨基酸序列,发现除了PeCIPK24b,都含有一个典型的丝氨酸／苏氨酸激酶区域,一个与CBL结合的NAF结构域及一个可能与磷酸酶相互作用的PPI结构域。而PeCIPK24b,可能是胡杨长期在高盐胁迫条件下的生境压力造成了激酶区域中一个关键的位点,即结合ATP的赖氨酸位点发生了突变。该位点由赖氨酸突变成了天冬酰胺,其激酶属性完全丧失。2.将PeCBL和PeCIPK分别构建至酵母双杂交载体中(pGBKT7和pGADT7),进行酵母双杂交实验。结果显示,PeCBL 1与PeCIPK3a、5、6b、7、9a、12b、15、16、23a、24a之间有相互作用。PeCBL4与PeCIPK23a、PeCIPK24a之间有相互作用。进一步通过BiFC手段验证胡杨PeCIPK23a、23b、24a、24b与10个胡杨PeCBL之间的相互作用关系发现,PeCIPK23a、23b与所有10个PeCBL之间都有相互作用；PeCIPK24a与PeCBL1、2、3、4、6、7、10之间有相互作用。由于在拟南芥中已经阐明与胡杨PeCIPK23a、24a同源的AtCIPK23、24分别介导低钾和高盐信号转导；所以我们推断,胡杨PeCBL1、PeCBL4可以通过与PeCIPK23a、24a相互作用形成多种不同的组合(complex),各组合之间相互影响、相互关联,与低钾、盐胁迫信号转导密切相关。3.通过转基因手段验证胡杨PeCBL1、PeCBL4、PeCIPK23a、PeCIPK24a在调控低钾、高盐胁迫下的信号转导机制表明,胡杨PeCBL1、PeCIPK23a可以互补拟南芥突变体因AtCBL1、AtCIPK23缺失所造成的对低钾胁迫的敏感性；PeCBL4、PeCIPK24a可以互补拟南芥突变体因AtCBL4、AtCIPK24缺失所造成的对盐胁迫的敏感性。与拟南芥一样,在胡杨细胞中,PeCBL1-PeCIPK23介导低钾胁迫的信号转导;PeCBL4-PeCIPK24介导高盐胁迫的信号转导。另外,我们发现PeCBL1虽然与PeCIPK24a有相互作用但是它们并不直接介导盐胁迫信号转导；PeCBL4与PeCIPK23a有相互作用,但是并不直接介导低钾胁迫的信号转导。4.我们对PeCIPK可能调控的下游一类Shake-like型钾离子通道进行了研究。酵母杂交和BiFC结果表明该家族中与拟南芥AKT1同源的两个成员PeKC1、PeKC2与PeCIPK23a有相互作用。向拟南芥野生型植株和突变体aktl中超量表达PeKCl和PeKC2能大大提高转基因植物对低钾胁迫条件的抗性。以上结果表明胡杨PeCBL1-PeCIPK23是通过磷酸化调控下游一类Shaker-like型钾离子通道PeKC1、PeKC2来实现低钾胁迫信号转导的。5.在毛果杨基因组中鉴定出了13个与真核生物内含子剪切有关的ECT基因。其中6个成员在胡杨中得到了克隆并将其构建至了酵母双杂交载体。该基因家族在拟南芥中发现受CBL-CIPK信号途径的调控。本研究为下一步研究该家族的信号调节及其它具体功能奠定了基础。总之,本研究通过生物信息、酵母杂交、BiFC及转基因等手段在胡杨中鉴定出了调控低钾胁迫的PeCBL 1-PeCIPK23a/b-PeKC 1/2信号途径及PeCBL4-PeCIPK24a调控的盐胁迫信号途径；这两个途径相互影响,共同调节低钾、高盐等胁迫的信号转导。我们的工作为今后进行抗逆相关的分子植物育种提供了重要理论基础。更多还原

【Abstract】 In plant, intracellular calcium ions as the second messenger play an important role in response to multiple environment stresses. The calcium binding proteins can sense the messenger and regulate the downstream responses. In plant, it has been identified many calcium binding proteins including CaM, CDPK and CBL. The CBL, as the plant calcium-binding proteins recently identified, are one group of small proteins. They must function by interacting with and regulating a group of serine-threonine protein kinases called CIPK and constitutes a complex of signal transduction pathways. In Arabidopsis, the CBL-CIPK pathway has been well studied. However, the molecular mechanism remains to be elucidated in woody plant. Due to its rapid growth, poplar becomes a model tree species with complete sequencing of the whole genome (Populus trichocarpa), which make it easy for identification the CBL-CIPK pathway in Populus. Here, we comprehensive functional characterized of the CBL-CIPK pathways in one stress-tolerant Populus species, Populus euphratica.1. We first identified 10 potential CBL and 25 CIPK genes in the Populus genome. Comparative genomics analyses in Populus and Arabidopsis showed that the two families appear to be much conserved in size and structure. However, compare with Arabidopsis, Populus have more paralogous gene pairs in genome. Therefore, we presumed these paralogous gene pairs might have shared a very recent duplication event in the poplar genome and the duplication events in Populus might have contributed to the expansion of the CBL family. To elucidate the functions of them, we cloned 10 CBLs and 14 CIPKs (PeCIPK3a,5,6b,7,9a, 11a,12b,14,15,16,23a,23b,24a,24b) from Populus euphratica. We investigated the expression patterns of CBLs in Populus euphratica under abiotic stress treatment. The results indicated that 7 PeCBL gene members (PeCBLl,2,3, 4,5,9, and 10) can be regulated in correspondence to specific external stress. By aligned with the amino acid sequences of each PeCBL, we found that the structure is rather conserved except in their N-termini regions, which may play an important functional role for protein sub-cellular location. For 14 PeCIPKs in Populus euphratica, except PeCIPK24b, we found that they all contain one kinase-domain, one NAF domain and one PPI domain. Because Populus euphratica has a long time to grow under high salt environment, we deduced the salt stress pressure maybe result in a key ATP-binding site in the kinase domain is mutated by the asparagines. That means the CIPK24b kinase completely lost its properties.2. We cloned the 10 PeCBLs into pGBKT7 vector and the 14 PeCIPKs into pGADT7, and then carried the yeast 2-hybrid experiment. The results showed that PeCBL1 can interact with PeCIPK3a,5,6b,7,9a,12,15,16,23a,24a; and PeCBL4 can interact with PeCIPK23a, PeCIPK24a. Also, we used the BiFC system to confirm the interaction between PeCIPK23a,23b,24a,24b and the 10 PeCBLs, and found that PeCIPK23a,23b can interact with all the 10 CBL; PeCIPK24a can interact with PeCBL1、2、3、4、6、7、10. Because in Arabidopsis, it has been demonstrated that AtCIPK23 and AtCIPK24 (homologous to PeCIPK23a/23b and PeCIPK24a/24b respectively) can interact with AKTl and SOS1 and regulate the low K+and salt stress pathway; we deduced that PeCBL1,4 can interact with PeCIPK23a/24a, forming different complex, and may play a crucial role in keeping the ion homeostasis in Populus euphratica.3. To further analyze the function of PeCBLl, PeCBL4, PeCIPK23a and PeCIPK24a in Populus euphratica to regulate low K+and salt stress, we over-expressed them in the mutant of Arabidopsis. It shows that PeCBL1、PeCIPK23a can complement the function of AtCBL1 and AtCIPK23 in cbll/9 and cipk23 mutant and they can recovery the mutant phonotype sensitive to low K+stress. PeCBL4、PeCIPK24a can complement the function of AtCBL4 and AtCIPK24 in cbl4 and cipk24 mutant and they also can recovery the mutant phonotype sensitive to salt stress. Otherwise, we find that PeCBL1-PeCIPK24a can’t mediate salt stress signal transduction and PeCBL4-PeCIPK23a can’t mediate low K+ stress signal transduction, though they can interact with each other.4. In addition, we investigated the candidate downstream genes of CBL-CIPK pathway, the shaker-like potassium channel family (KC). The results of yeast-2 hybrid and BiFC showed that PeKC1 and PeKC2, homologous with AKT1, can interact with PeCIPK23a. The transgenic plant of PeKCl or 2 can greatly improve the resistance to low K+stress. The result indicated that PeKC1 and PeKC2 are involved in the PeCBL1-PeCIPK23 signal transduction and play an important role under low K+stress. 5. We also indentified 13 ECT containing a splicing factor (YTH) domain from Populus genome and six of them from Populus euphratica have been clone into yeast-2 hybrid vector. In Arabidopsis, it has been demonstrated the ECT proteins may play a critical role in relaying the CBL-CIPK signals, thereby regulating gene expression. The work will lay a foundation for further study on concrete function of the gene family.Taken together, the study has clearly identified two signal transduction pathways in Populus euphratica by biological information, yeast 2-hybrid, BiFC, transgenic method and so on. One is the PeCBL1-PeCIPK23a/23b-PeKC1/2 pathway, and the other is the PeCBL4-PeCIPK24a pathway. And also we found that the two pathways can rergulate each other and play an important role in corresponding to low K+and salt stress. Our results will provide an important foundation for further carrying molecular plant breeding to improve stress resistance in future.更多还原