Cloning And Functional Analysis Of TaCBLs And TaCIPKs Genes In Wheat

CBLs, a new type of Ca²⁺ sensors unique to higher plants, called for their similarity to B-subunit of yeast and animal calcineurin, could monitor the instant changes of Ca²⁺ concentration and stimulate the downstream members on signaling pathway then trigger plant physiological effects. Though capable of binding Ca²⁺, CBL must interact with its the target protein CIPKs to perform the functions. CIPKs（CBL-interacting protein kinases）, unique to plants, consist of a conserved N-terminal kinase domain and a C-terminal regulatory domain. The CIPK catalytic domain harbors a typical activation loop with a threonine residue located between the N-terminal and C-terminal amino acid motifs DFG and APA, respectively. Within the C-terminal regulatory domain of the CIPKs, a stretch of 21²4 amino acids designated as the NAF domain was identified as a novel interaction module sufficient and required to mediate the interaction with the CBL proteins. Adversity such as soil salinization, drought, low temperature, high p H and low K+ is a major environmental limiting factor in plant growth and development. The CBL-CIPK pathway is emerging as a major calcium signaling network in plants. The Ca²⁺-CBL-CIPK signal system is capable of sensing stress and transmitting stress signals, thereby regulating the specific cellular biochemical processes. Studies on the interaction of CIPKs and CBLs family may identify the molecular mechanisms of stress responses and other stimulus-response coupling processes in plants.At present, the study about CBL-CIPK network is mostly restricted to Arabidopsis and rice, there are few researches about wheat. Wheat is one of the world’s most important food crops, stresses such as drought, low temperature, salinization seriously affect wheat growth and yield, searching sress-resistence related genes and putting into use is is one of the effective ways to improve wheat resistance to adversity stress. CIPKs in plants have an important role in answering some environmental stresses signals and adapting to environmental changes. In order to reveal the structural and functional characteristics of Ta CBLs and Ta CIPKs in the molecular level, and provide the guidance for the research and application to wheat breeding, we cloned several genes including Ta CBL1, Ta CBL2, Ta CBL6, Ta CBL7, Ta CIPK9, Ta CIPK15, Ta CIPK23 and Ta CIPK31. We analyzed the expression characteristics of Ta CIPKs genes according to Real Time PCR. The interactions between Ta CBLs and Ta CIPKs were detected by yeast two-hybrid system. In brief, the main results were summarized as below.1. To better understand the role of CBL-CIPK in stress-responsive pathway, we firstly cloned the Ta CBLs and Ta CIPKs gene using RACE and RT-PCR. The coding region of Ta CBL1, Ta CBL2, Ta CBL6 and Ta CBL7 is 648 bp, 678 bp, 681 bp and 642 bp, respectively. Ta CBL1 is predicted to encode a protein of 215 amino acids, Ta CBL2 is predicted to encode a protein of 225 amino acids, Ta CBL6 encodes 226 amino acids and Ta CBL7 encodes 213 amino acids. Ta CIPK9, Ta CIPK15, Ta CIPK23 and Ta CIPK31 encodes 443, 432, 453 and 449 amino acids, respectively.2. Amino acid sequence homology analysis indicated Ta CBL1, Ta CBL2, Ta CBL6, Ta CBL7 are highly homologous proteins and Ta CIPK9, Ta CIPK15, Ta CIPK23, Ta CIPK31 are highly homologous proteins, too. Functional domain prediction suggested proteins encoded by four Ta CBL genes all contained four EF-hand structures highly conservative in different protein, and each EF-hand is consisted of 12 relatively conservative amino acids. Proteins encoded by Ta CIPK genes belong to serine-threonine protein kinase family and harbor N-terminal kinase domain and C-terminal regulatory domain. C-terminal regulatory domain is partitioned into the two interaction domains NAF domain, which is responsible for CBL-CIPK interaction and PPI domain for interacting with PP2 C.3. Expression characteristic analysis suggested that the gene expression of Ta CIPK9, Ta CIPK23 and Ta CIPK31 was up-regulated in wheat root under salt-sress condition, while only Ta CIPK15 was down-regulated expressed. In leaf, Ta CIPK9 expression was up-regulated. Under low temperature treatment, we observed that the changing trend of four Ta CIPKs was consistent, their expression in root was up-regulated, but they were all down-regulated expressed in leaf. During 100 μM ABA treatment, Ta CIPK9 and Ta CIPK15 were up-regulated expressed and the expression of Ta CIPK23 and Ta CIPK31 rised first then declined in root. In leaf, the expression of Ta CIPK15 and Ta CIPK31 appeared to be up-regulated, while Ta CIPK9 and Ta CIPK23 showed weaker expression. These results demonstrated that Ta CIPK9, Ta CIPK15, Ta CIPK23 and Ta CIPK31 genes may be involved in multiple stress response pathways in plants.4. In Yeast Two-Hybrid assay, the positive control p GBKT7-53×p GADT7-T and the diploids BD-Ta CBL1×AD-Ta CIPK9, BD-Ta CBL1×AD-Ta CIPK15, BD-Ta CBL1×AD-Ta CIPK23, BD-Ta CBL2×AD-Ta CIPK15, which were fused by the transformed Y187 yeast strain and the transformed Y2 HGold yeast strain, grew on selectiveagar plate（SD/-Ade/-His/-Leu/-Trp/X-α-Gal/Ab A）, and the resulting colonies were light blue, the negative control p GBKT7-lam×p GADT7-T couldn’t grow on selective agar plate（SD/-Leu/-Trp/X-α-Gal/Ab A）. The assay results strongly indicated that Ta CBL1 could interact with Ta CIPK9, Ta CIPK15 and Ta CIPK23, and Ta CBL2 could interact with Ta CIPK15. This provided a basis for research on interactions and interaction sites of Ta CBLs and Ta CIPKs protein thus lay the foundations for development of CBL-CIPK signaling network.In summary, cloning and functional analysis of Ta CBLs and Ta CIPKs genes provide basic information for studying the role on wheat growth and development under adverse conditons and help us to better understand the molecular mechanisms of calcium signaling during stress responses.