Analyses On The Evolution Of Plant CaM/CML Gene Family And Its Structural And Expressional Characteristics In Nelumbo Nucifera

As a ubiquitous second messenger in organism,Ca²⁺and its signal were mainly detected and decoded by its key sensors calmodulin（Ca M）and calmodulin-like（CML）proteins,which occurs in a wide range of cellular processes such as plant growth and development,and stress response.So far,the studies of Ca M/CML family are mainly focused on the functional analysis rather than the origin and evolution.Meanwhile,Ca M/CML genes have not been identified in sacred lotus（Nelumbo nucifera）,a basal eudicot and important horticultural plant,and the structural and expressional characteristics of these genes are yet to be elucidated in this species.With the rapid development of high-throughput sequencing,more and more plants’genomes have been sequenced and assembled.The 1KP project offers a variety of transcriptomic data from algae to angiosperms,and the genome of Nelumbo nucifera var.China Antique has been sequenced and assembled.Therefore,this investigation is designed based on the unparalleled resource and bioinformatic tools to analyze the evolution of the Ca M/CML gene family in plants and the structure,function and expression pattern of the Ca M/CML gene family in sacred lotus.The main research contents and results are as follows.1.A total of 25227 Ca M/CML proteins were identified in 1230 plant species through BLASTP and HMMER analyses,including 2133 Ca M and 23094 CML genes.All the analyzed species at each stage from algae to angiosperms contain this gene family.It was found that the Ca M was originated in Green algae and the CML was originated in Dinophyceae,which indicates that CML appeared earlier than Ca M in plants.Furthermore,it seems that the expansion of CML gene family is correlated with genome duplication events.2.The phylogenetic trees were constructed based on the protein sequences of Ca M/CML,which could divide this gene family into seven groups.The results showed that sequence of Ca M was conserved and the Ca M from plants at the same evolutionary stage tended to cluster together.However,CML was diverse and the CML in algae was always located at the base of the phylogenetic tree branches.Besides,the high expression genes were clustered in the base of the tree and were conserved,while the low expression genes were diverse.3.The analysis of the conserved domain showed that the EF-hand domain Ca M contains three D-x-D-x-D motifs and E-x₂-E motifs,while CML contained two D-x-D-x-D motifs in the fifth and sixth EF-hand domain.In terms of sequence characteristics of the EF-hand domain,Ca M is more conserved than CML,and the EF-hand domain contained in CML became more diverse with the evolution of plants.4.Based on the genome-wide identification,thirty-four Ca M/CML genes were identified in sacred lotus.Phylogenetic analysis showed that they could be divided into nine groups.Gene structure and conserved motif analyses demonstrated the conservation and divergence of Ca M/CML in sacred lotus.Cis-acting elements analysis indicated that they might be related to plant growth and development,abiotic stress,and plant hormones.In addition,expression analysis showed that Ca M/CML was differentially expressed in various tissues.Meanwhile,the expression patterns of Nn Ca M/Nn CML under the treatment of Ca Cl₂ and EDTA were analyzed by q RT-PCR,and the result showed that 14 Nn Ca M/Nn CML genes responded to calcium treatments in roots.5.Weighted gene co-expression network analysis of public transcriptome data of lotus wild variety“China Antique”with different tissues presented the expression connectivity of Nn Ca M/Nn CML,which were divided into 11 modules.Gene ontology analysis of the genes in each module demonstrated that Nn Ca M/Nn CML may be involved in extensive biological processes,such as synthesis and processing of DNA and RNA,and protein post-translational modification and each module specifically correlated with the phenotypes including the development of leaves’petiole and lotus seed,implying the wide regulation of Nn Ca M/Nn CML in lotus.Taken together,this study applied bioinformatics,phylogenetics,and molecular biology methods to study the origin and evolution of the Ca M/CML gene family in plants,and the expression pattern of the Nn Ca M/Nn CML gene family in different tissues and calcium treatments.This study will deepen the understanding of the relationship between the evolution of the Ca M/CML gene family and the phylogeny in green plants,and provided a theoretical basis for exploring the functional differentiation of the Ca M/CML gene family in the process of species evolution.Meanwhile,our results will enhance the understanding and lay a foundation for further study on the functions of Ca M/CML in lotus.