Function Analysis Of TaNADK1/2 And Its Interaction With TaCaMs In Wheat (Triticum Aestivum L.)

Calcium(Ca²⁺)signaling and nicotinamide adenine dinucleotide（NAD）signaling are two basic signal regulation pathways in organisms,playing crucial roles in signal transduction,energy metabolism,stress tolerance and various developmental processes of cells.Notably,calmodulins（CaMs）and NAD kinases（NADKs）are important hubs for connecting these two types of signaling networks,where CaMs are the unique activators of NADKs.By using the bioinformatics and molecular biology methods,we analyzed the basic functions of TaNADK1/2 and preliminarily proved that CaM could interact with NADK in wheat.These specific research results are as follows:1.Expression patterns of wheat TaNADK1 and TaNADK2 genes were analyzed by qRT-PCR.The results of tissue expression pattern indicated that these two genes are specifically expressed in various tissues.TaNADK1 is mainly expressed in panicles,while TaNADK2 is mainly expressed in leaves and roots.Inducible expression pattern showed that both TaNADK1 and TaNADK2 are responsive to ABA,MeJA,IAA and MV treatments.In addition,the TaNADK1/2 overexpression vectors were constructed and the transgenic Arabidopsis overexpressing lines were obtained through the floral dip method.The SWISS-MODEL,western blot and split-luciferase complementation results revealed that TaNADK1/2 are mainly present as multimers in plants.2.A total of 40 CaM family genes were genome-wide identified from hexaploid bread wheat database（Triticum aestivum L.）by blasting with these known and typical CaM sequences.According to the phylogenetic tree of the TaCaMs and their gene structure and motif organization,we divided these TaCaM genes into six subfamilies.By selecting the representative members in each of the six subfamilies,namely TaCaM1-1A,TaCaM2-1B,TaCaM4-2A,TaCaM5-3A,TaCaM9-4B,and TaCaM12-5A,the subcellular localization of these TaCaMs was analyzed and the results showed that all of the six selected TaCaMs are localized at the cytoplasm,nucleus and plasma membranes.3.TaNADKs and TaCaMs are fused to N-and C-terminally of luciferase（nluc and cluc）,respectively.And we then checked the possible interaction relationships of these TaCaMs with the two TaNADKs by using a split luciferase system in tobacco leaves.The results showed that TaCaM1/2/4/5/9 could bind to TaNADK1/2,while TaCaM12-5A could not.In conclusion,through the analysis of the expression patterns of TaNADK1 and TaNADK2 genes in wheat,the proof of TaNADK1 and TaNADK2 polymer forms,and the creation of TaNADK1/2 overexpressing Arabidopsis transgenic lines,our study laid a theoretical foundation for the further study of TaNADK1/2 functions.By genome-wide identification of TaCaM family and the proof of the interaction between TaCaMs and TaNADKs,this study also provides an important reference for the in-depth understanding of CaM and NADK and their regulatory mechanisms in wheat.