A Mongolian Pine Specific Calmodulin-like Calcium Binding Protein Physiological Function Research

Many wild plants carry genes such as insect resistance and drought resistance,which are important for improving crop varieties and increasing the diversity of crops.Many wild plants are a"gene pool"for agricultural breeding.High-throughput and low-cost DNA and RNA sequencing technologies greatly facilitate the identification of these genes.Here,we conducted a transcriptional analysis of Pinus sylvestris var.mongolica Litv.,an evergreen pine in northeastern China,to identify a novel CALMODULIN-LIKE protein-encoding gene,PsCML1,no significant homologs found in the NCBI protein database.The main experimental results on these aspects are as follows.?1?PsCML1 encodes a protein predicted to have a single trans-membrane domain at its N-terminal.Four EF-hand motifs(calcium[Ca²⁺]-binding structures)are located at its C-terminal and showed Ca²⁺-specific affinity in isothermal titration calorimetric?ITC?analysis.?2?Transient expression of PsCML1 in Nicotiana benthamiana showed that the PsCML1 localizes to the endoplasmic reticulum?ER?.?3?Heterologous expression of PsCML1 in Arabidopsis significantly promoted seedling growth and increased resistance to salt stress and Ca²⁺deficiency.Using with a laser confocal microscope revealed that there have significant difference in length of root apical meristem?RAM?and cell numbers between PsCML1 transgenic plants and wild type.Ps CML1 transgenic plant have higer RAM and more meristem cells than that of the wild type.However,there are not significant difference in average cell length of RAM.This means that the PsCML1 transgene has the ability to enhance growth of root.?4?The cell wall components biosynthesis linked with protein glycosylation in the ER and reactive oxygen species?ROS?homeostasis.Through DHR staining,we found that the transgenic roots had higher steady-state levels of H₂O₂ than those of the WT.There is no significant difference was found in the extent of protein glycosylation through between the transgenic and WT plants by immunoblotting.Our research provid theory basis for studing resistance genes from wild plants.