棉属野生种旱地棉蛋白激酶基因GarCIPK8的克隆与功能分析

Isolation and Functional Analysis of GarCIPK8 Gene from Gossypium aridum

CIPK(calcineurin B-like calcium sensor interacting protein kinase)是植物钙感受器钙调磷酸酶B类似蛋白特定靶向的一类丝氨酸/苏氨酸蛋白激酶。在前期的研究中,我们将棉属野生种D亚组旱地棉(Gossypium aridum)盐胁迫前后RNA混合样品进行转录组测序,发现一CIPK相关基因存在较高的转录丰度。盐胁迫下荧光定量PCR分析表明该基因在根部表现诱导上调表达,推测该基因可能参与植物在高盐胁迫下的生理调控。利用电子克隆及RT-PCR方法从旱地棉中克隆了一个ORF全长为1350bp的蛋白激酶基因GarCIPK8。序列分析表明该基因编码的蛋白含有449个氨基酸,分子量为51.12kD,理论等电点为8.13,其N端催化结构域包含一个丝氨酸/苏氨酸蛋白激酶域,C端具有植物CIPK蛋白家族特有的24个氨基酸组成的NAF保守结构域;与水稻OsCIPK8蛋白的相似度为73.95%。为进一步验证其功能,利用组成型高效强启动子CaMV35S和拟南芥逆境胁迫启动子rd29A构建植物表达载体并转化烟草,经PCR和RT-PCR分子检测,证明GarCIPK8基因已经整合到烟草基因组中,并正常表达。T1代转基因植株耐盐性鉴定结果表明GarCIPK8基因对提高转基因植株的耐盐性有较明显的作用,PEG干旱模拟试验也证明GarCIPK8基因可增强植株的抗旱性。

CIPK （calcineurin B-like calcium sensor interacting protein kinase） is a typical group of serine / threonine protein kinase targeting calcium sensor calcineurin B-like protein-specific. In a previous study, we obtained the transcriptome of Gossypium aridum with a pooled RNA sample from root and leaf of G aridum under salt stress. From the transcriptome data, we founded that a C1PK related gene had high level of expression abundance. Real-time fluorescence quantitative PCR analysis showed its relative expression level was up-regulated in root induced by salt stress, indicating this CIPK related gene might be involved in response to salt stress during cotton seedling stage. By in silico cloning and RT-PCR technology, a full-length cDNA encoding a CIPKs homologue （GarC1PK8） was isolated from G. aridum. The deduced GarCIPK8 protein had 449 amino acids with a predicted molecular weight of 51.12 kD and a predicted isoelectric point of 8.13. The kinase N-terminal catalytic domain of GarCIPK8 exhibited a typical serine / threonine protein kinase domain. The conserved 24 amino acid motif existed in C-terminal non-kinase regions of GarCIPK8 protein. Alignment of amino acid sequence showed that GarCIPK8 was 73.95% identical to Oryza sativa OsCIPK8 protein. In order to characterize its putative function, the GarCIPK8 gene driven by CaMV35S promoter and stress-specific promoter rd29A respectively were transformed into tobacco by Agrobacterium-mediated transgenic technology PCR and RT-PCR detection confirmed that GarC1PK8 gene was integrated into tobacco genome and expressed normally. The growth of transgenic plants of T1 generation was observed under salinity and drought stresses, showing that transgenic plants en- hance tolerance to both high salt and osmotic stresses.