资源植物与环境工程研究组知识库

钙离子依赖的蛋白激酶(CDPKs)是一类钙离子结合蛋白，在生物体钙离子信号转导途径中发挥着重要作用。该类激酶广泛存在于植物界，并且在部分原生生物中也有发现。CDPKs是目前植物中研究较为深入的一类激酶，在拟南芥和水稻中，该基因家族已经得到了系统鉴定与分析。已有的研究表明，相当一部分的CDPK基因参与植物胁迫响应。然而，在杨树这一模式树种植物中，对CDPK基因家族的研究尚未有报道。

本研究对杨树(Populus trichocarpa)的CDPK及其密切相关的基因家族进行了系统性的分析，共鉴定出了30个CDPK基因家族成员和20个与CDPK密切相关的成员，这20个成员分属4个基因家族，包括CDPK相关激酶家族(CRK)，钙和磷酸烯醇式丙酮酸羧化酶激酶家族(PPCK)，PPCK相关激酶家族(PEPRK)以及钙和钙调素依赖的蛋白激酶(CCaMK)家族。对鉴定的杨树CDPK及其相关基因家族成员进行染色体定位后发现，这些基因较为分散地分布在15个杨树的遗传连锁群上。聚类分析证实这些基因共同形成了CDPKI-IV、CRKs、PPCKs、PEPRKs和 CCaMKs 8个较为独立的进化分支。通过进一步的剪切位点分析，我们推测这些激酶可能具有相同的起源。杨树CDPK基因家族内部共有14对同源基因对，而在其相关的激酶家族中则发现了8对同源基因对，其中，12对CDPK基因对和全部8对相关激酶基因都对定位在杨树基因组的复制区并且在进化过程中被保留。在这些基因的保留和维持中，纯化选择(purifying selection)发挥了主要的作用。为了分析CDPK及其相关基因在杨树不同组织及不同胁迫条件下的表达情况，我们利用生物芯片进行了一系列的表达谱分析，发现某些基因的表达具有明显的组织特异性，同时部分基因在特定的胁迫条件下表达量明显上调或下调，由此预测了部分可能与杨树胁迫响应有关的基因；另外对同源基因对的表达模式进行分析后，发现不同基因对经历了不同的进化命运。最后，为了验证可能参与干旱胁迫响应的CDPK基因，我们将杨树进行干旱胁迫处理不同时间后，利用实时荧光定量PCR(RT-qPCR)检测了9个候选基因的表达情况，结果发现8个基因的表达明显受到干旱胁迫的诱导。以上研究为进一步揭示杨树CDPK及其相关激酶家族的生物学功能奠定了基础。

Elongator复合物是一种参与转录延伸的组蛋白乙酰转移酶复合物，ELP1为Elongator复合物中最大的亚基。在拟南芥中，突变体elp1叶片卷曲，根系变短，花青素大量积累，并出现抗旱及对ABA高度敏感等性状。表明其可能在植物ABA信号传导通路和干旱响应过程中具有重要的作用。

本研究克隆得到了杨树PtELP1基因，对其进行了系统进化和蛋白结构预测等生物信息学分析。并且利用半定量PCR检测了PtELP1的组织表达情况，同时通过亚细胞定位实验证明PtELP1定位在细胞核与叶绿体上。为了进一步验证其功能，我们构建了PtELP1的过表达载体，并将其分别转化野生型拟南芥和突变体elp1，得到了过表达和互补的转基因植株。通过表型观察及生理生化实验发现，虽然过表达植株与野生型相比没有明显区别，但PtELP1几乎可以完全互补突变体elp1的各种表型，不再出现抗旱或ABA高度敏感等性状。以上实验证明PtELP1为拟南芥ELP1的功能同源基因，并且参与ABA信号传导通路和干旱响应的过程。

Calcium-dependent protein kinases (CDPKs) are Ca²⁺-binding proteins identified throughout plant kingdom and in certain types of protists which known to play crucial roles in Ca²⁺ signal transduction pathways. Although Genome-Wide analysis of CDPKs had been carried out in Arabidopsis and rice, and quite a subset of CDPKs were proven to play crucial roles in plant stress responsive signature pathways, no study has been conducted in model tree species Populus thus far.

In this study, a comprehensive analysis of Populus (Populus trichocarpa) CDPK and its closely related gene families was performed. Thirty CDPK genes and twenty closely related kinases genes encoding CDPK-related kinases(CRK), calcium and phosphoenolpyruvate carboxylase kinases (PPCKs), phosphoenolpyruvate carboxylase kinase-related kinases (PEPRKs) or calcium and calmodulin-dependent protein kinases (CCaMKs) respectively were identified in Populus genome.

These genes were phylogenetically clustered into eight distinct subfamilies (CDPKI-IV, CRKs, PPCKs, PEPRKs and CCaMKs) and predominately distributed across fifteen linkage groups (LGs). Analysis of the splicing sites revealed that these kinases are originated from a common ancestor. Twelve genes out of fourteen Populus CDPK paralogous pairs and all of the eight closely related gene paralogous pairs were located in the duplicated blocks of Populus genome and then preferentially retained during the sequential evolutionary courses. Genomic organization analyses indicated that purifying selection has played a pivotal role in the retention and maintenance of Populus CDPK gene family. Microarray analysis showed a number of Populus CDPK and its closely related genes differentially expressed across different tissues and under various stresses, according to which a sub set of putative stress responsive genes were proposed. The expression profiles of paralogous pairs revealed they might undergo different evolution fates after segmental duplications. Finally, in order to verify the expression profile of putative drought-responsive Populus CDPK genes, plants were exposed to drought stress for different times and quantitative real-time RT-PCR (RT-qPCR) was performed on 9 selected genes.

Taken together, these observations may lay the foundation for future functional analysis of Populus CDPK and its closely related gene families to unravel their biological roles.

ELP1 is the biggest subunit of Elongator, which is a histone acetyl-transferase complex. In Arabidopsis, mutants elp1 had narrow leaves, reduced root growth, an increased accumulation of anthocyanins, ABA hypersensitivity and was more tolerant to drought stress, which indicating that ELP1 might be involved in the ABA signaling pathway and response to drought stress.

In the present study, we obtained the PtELP1 gene from Populus and analyzed its systematic phylogeny and protein structure. The tissue-specific expression was investigated by RT-PCR and the subcellular location experiment was performed which revealed PtELP1 was located in the nucleus and chloroplast. In order to confirm its biological functions, vector overexpressing PtELP1 was constructed and transformed into wild type Arabidopsis and mutant elp1, by which the PtELP1-overexpressing and completing transgenic plants were both obtained. According to the phenotype and physiology analysis, although transgenic plants overexpressing PtELP1 showed no difference with the wild type plants, overexpression of PtELP1 in mutant elp1 rescued most of the changes, including morphological changes, ABA hypersensitivity and tolerance to drought stress. The results above demonstrated that PtELP1 was functional ortholog of ELP1 in Arabidopsis and played crucial roles in modulating ABA and drought stress responses.