Characterization Of The TaCBLs And TaCIPKs Genes In Wheat And Interaction Analysis By Yeast Two-hybrid System

Ca²⁺ is a universal plant in the second messenger,its sensor calcineurin CBL（Calcineurin B-like protein）and the interaction of protein kinase CIPK（CBL-interacting protein kinase）to form Ca²⁺-CBL-CIPK network signaling pathway,which play an important role in plant growth and development in the process of stress response.The CBL protein family which has four conserved EF hand structures,each of which is composed of 12 amino acids,which is bound to Ca²⁺ harbor a typical loop structure.The activity of the ring between N-terminal kinase domain incluing amino amino acid DFG and APE terminal kinase domain of CIPK protein（activation loop）,can be used as target sites for other protein kinase phosphorylation,C-terminal region of CIPK contains a unique combination of 24 amino acid regulatory domain,namely NAF or FISL domains of CIPK kinase the activity of inhibition.The concentration of Ca²⁺ in a certain concentration,NAF/FISL and CBL combined,which can relieve the inhibition of CIPK kinase region,so that CIPK kinase into the activated state.Wheat is the most widely planted,the highest total yield of grain crops in the world,but the low temperature,drought,salinity,high p H value and low potassium and other abiotic stresses have seriously affected the yield and quality of wheat,and the Ca²⁺-CBL-CIPK network signaling pathway of these stress has certain inhibitory effect.Study on the regulation of Ca²⁺-CBL-CIPK network in wheat is far behind of Arabidopsis and rice,in order to further understand the molecular mechanism of the structure and function of Ta CIPK and Ta CBL in wheat,by homologous cloning of genes Ta CIPK and Ta CBL family,analysis of the biological information;using fluorescence quantitative PCR（Real-time PCR）.Method for analysis of Ta CIPK gene in stress specific expression and temporal specific expression;the yeast two hybrid（Yeast Two-hybrid System）method to verify the interaction between Ta CIPKs and Ta CBLs control network.The main results are as follows:Ta CBL3,Ta CBL9,Ta CIPK3 and Ta CIPK11 genes were cloned from wheat by homologous cloning method.The coding region of Ta CBL3,Ta CBL9,Ta CIPK3 and Ta CIPK11 is 678 bp,1677 bp,and 1524 bp,respectively.Ta CBL3 is predicted to encoding 225 amino acids;Ta CBL9 is predicted to encoding 296 amino acids;Ta CIPK3 is predicted to encoding 447 amino acid residues with a molecular mass of 50.5 k D and an isoelectric point of 8.6;Ta CIPK11 is predicted to encoding 507 amino acid residues with a molecular mass of 57.5 k D and an isoelectric point of 8.75.Homology analysis showed that the gene of the Ta CBL family of wheat was higher than that of the family genes such as Arabidopsis,rice,short stalked grass,barley,and so on.The results showed that the amino acid sequence of gene was high.The homology of Ta CBL9 and wild barley Hb CBL9 and rice were 78% and 74%,respectively.The homology of Ta CBL3 and Hv CBL3,Zm CBL3 and Os CBL3 of rice was Os CBL9,respectively,to 96%,94%,94%.Functional domain analysis showed that Ta CBL3 and Ta CBL9 gene encoding the protein contained 3 typical EF-hand structure in different proteins in their conservation are high,and each EF-hand consists of 12 conserved amino acid composition.Amino acid sequence homology analysis indicated Ta CIPK3,Ta CIPK11 are highly homology protein,similarity between Ta CIPK3 and Ae CIPK31,the Ural wheat Aegilops Tu CIPK31 and Brachypodium Bd CIPK31 were as high as 100%,96%,90%;Ta CIPK11 and Ae CIPK11,the Ural wheat Aegilops Tu CIPK11,Brachypodium Bd CIPK11,barley Hv CIPK11 and the similarity were respectively 97%,97%,90%,89%.The Ta CIPK3 and Ta CIPK11 show that the regulatory domain of wheat protein kinase domain containing N-terminal and C-terminal,comprising a protein kinase activation loop（Activation loop）and NAF/FISL motif structure,used in combination with Ta CBL,which belongs to the serine/ threonine protein kinase family.Expression characteristic analysis suggested that the gene expression of Ta CIPK3 and Ta CIPK11 in 200 m M Na Cl（high salinity）,ABA（100μM）,4 ℃（low temperature stress）treatment of quantitative analysis,the results showed that Ta CIPK3 was up-regulated in root and leaf which induced by high salt and ABA treatment;low temperature induced processing in the roots of Ta CIPK3 was up-regulated,and the expression of Ta CIPK3 was down regulated in leaves.High salt induced Ta CIPK11 was up-regulated in the leaves,the expression of Ta CIPK11 was down regulated in roots;ABA induced treatment Ta CIPK11 was up-regulated in roots and leaves;low temperature induced Ta CIPK11 in the root and leaves of Ta CIPK11 were up-regulated.In a Matchmaker GAL4-based two-hybrid assay,DNA-p GBKT7 can identify GAL4 located upstream activation sequence and combined with the UAS,while the p GADT7-T is started by the UAS downstream gene transcription and the two kinds of fusion protein were expressed in yeast,if these two proteins are detected between interactions,p GADT7 and p GBKT7 will form a complete transcription the activation factor,and activation of the corresponding gene expression.Using yeast two hybrid-system,Ta CIPK3,Ta CIPK11 and Ta CBLs to verify the interaction of regulatory networks in yeast two hybrid system,Ta CBL1/2/3/4/6/7/9 was inserted into the DNA-p GBKT7 vector.The construction of p GBKT7/ Ta CBL1/2/3/4/6/7/9 fusion protein;Ta CIPK3/11 cloned into the p GADT7-T vector,to construct the p GADT7/ Ta CIPK3/11 fusion protein.The yeast two hybrid experiment in the positive control of p GBKT7-53 × p GADT7-T,Y187 and Y2 HGold fusion yeast（p GBKT7-Ta CBL1 × p GADT7-Ta CIPK3,p GBKT7-Ta CBL2 × p GADT7-Ta CIPK3,p GBKT7-Ta CBL3 × p GADT7-Ta CIPK3,p GBKT7-Ta CBL4 × p GADT7-Ta CIPK3,p GBKT7-Ta CBL2 × p GADT7-Ta CIPK11,p GBKT7-Ta CBL3 ×p GADT7-Ta CIPK11,p GBKT7-Ta CBL6 × p GADT7-Ta CIPK11,p GBKT7-Ta CBL9 × p GADT7-Ta CIPK11）to growth medium SD/-Ade/-His/-Leu/-Trp/X-alpha-Gal/Ab A in the selective solid,and the colony was light blue,while the negative control group Y187 and Y2HGold（p GBKT7-lam×p GADT7-T）SD/-Leu/-Trp/X-in-Gal/Ab A culture medium can’t grow.The results showed that Ta CBL1,Ta CBL2,Ta CBL3,Ta CBL4 and Ta CIPK3 could interact with each other.Ta CBL2,Ta CBL3,Ta CBL6 and Ta CBL9 could interact with Ta CIPK11 gene.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.