Identification Of Choline Kinase-Related Genes And Functional Verification Under Cd²⁺ Stress In Cotton

Choline kinase（CK）gene plays an important role in plant growth,development and in response to Cd²⁺stress.Its main function is to regulate the biosynthesis of phosphatidylcholine in cell membranes.This study aims to explore the relationship between the structure and function of CK genes,and to provide a framework for the study of the functions and biochemical properties of CK genes in various species under Cd²⁺stress.The results showed that most GhCK genes were affected by abiotic stress.Under Cd²⁺stress,the expression levels of GhCK gene family members changed to different degrees.The expression level of GhCK5 was significantly up-regulated,indicating that GhCK5 plays an important role in cotton tolerance to Cd²⁺stress.This study will further explore the molecular mechanism of GhCK5 gene in cotton tolerance to Cd²⁺through the evolution analysis of CK gene family and the functional verification of GhCK5.（1）Three-leaf stage Gossypium hirsutum cv Han 242 was treated with 4 m M Cd²⁺for 0 h,3 h,6 h,9 h,12 h,and 24 h.Compared with the control,cotton leaves showed the phenotype of water loss,leaf veins and stem base blackening.（2）Through RNA-seq analysis of cotton treated with 4 m M Cd²⁺for 9 h,the Cd²⁺-resistant gene CK gene be found in the glycerophospholipid metabolic pathway.The choline kinase gene family was analyzed by methods such as identification of CK gene family members,chromosomal location analysis and gene structure analysis.A total of87 CK genes were identified in 4 cotton species and 7 other diploid plants,of which 13genes were identified in Gossypium hirsutum.Chromosomal mapping results showed that 7 members were located on the At subgenome,and the other 6 members were located on the Dt subgenome.Cotton seedlings were treated at the three-leaf stage with cold,heat,salt,and drought stresses,and found that GhCK family members were differentially expressed to varying degrees,thus verifying that GhCK family members were involved in the regulation of abiotic stress.Under Cd²⁺stress,the up-regulated expression of GhCK5 was the most significant.（3）After the GhCK5 gene was silenced by VIGS（virus-induced gene silencing）experiments,the content of phosphatidylcholine in cotton leaves was significantly reduced.After the silenced and control plants were treated with 4 m M Cd²⁺,although the content of phosphatidylcholine in the silent plants increased significantly,it was still significantly lower than that in the control plants,and the growth status of the silent plants was significantly worse than that of the empty plants.The silenced plants appeared faster than the blank vector-injected plants with wilted leaves,blackened leaf veins,and blackened stem bases,and the enhancement of SOD activity was inhibited,at the same time,the chlorophyll content decreased.GhCK5 transgenic Arabidopsis thaliana was obtained through agrobacterium-mediated Arabidopsis thaliana infection experiments.The study found that lower concentration of Cd²⁺had no significant effect on the germination of WT and transgenic Arabidopsis thaliana,but the growth of WT was significantly worse than that of transgenic Arabidopsis thaliana.Under higher concentration of Cd²⁺stress,the germination rate of Arabidopsis thaliana was significantly worse lower,the germination rate of transgenic Arabidopsis thaliana was significantly higher than that of WT.The results of subcellular localization showed that the GhCK5 protein was mainly located in the cell membrane,which further verified that the GhCK5 gene played a role in the cell membrane.In summary,Choline kinase gene can help cotton to improve Cd²⁺tolerance by regulating the synthesis of phosphatidylcholine in the cell membrane.