Cloning And Functional Analysis Of RtCML42 And RtCDPK16 Regulatory Genes In Rare Salt-producing Plants Reaumuria Trigyna

Calcium,as an important second messenger in plant cells,not only regulates a variety of physiological activities in plant cells,but also resists external stress by activating some proteins or enzymes to regulate changes in calcium concentration in the cytoplasm,thereby triggering a response.Reaumuria trigyna belonging to the family Tamarieaceae?Reaumuria?,is a Tertiary ancient Mediterranean relic plant with strong xerophytic halophyte shrubs.It is found in East Alxa-West Ordos.In this study,based on transcriptome sequencing analysis,the cDNA of the RtCML42 and RtCDPK16 genes were successfully cloned,and construct eukaryotic expression vector Arabidopsis thaliana,through the study of its expression characteristics and gene function,to explore the role of RtCML42 and RtCDPK16in abiotic stress,and further analyze the mechanism of calcium signal regulating plant response to adversity.The main findings are as follows:1.Bioinformatics analysis showe,the ORF Length of RtCML42 is 657bp and encodes 218 amino acids.And the ORF of RtCDPK16 gene is 1689 bp in length and encodes 562 amino acids.Both proteins are hydrophilic proteins and have 4EF-hand binding to Ca²⁺.And RtCML42 has the highest homology with CBL3 of castor;RtCDPK16 has the highest homology with grape CDPK16.2.qRT-PCR analysis show,the expression of RtCML42 and RtCDPK16 can be induced by high temperature,low temperature,NaCl,PEG,ABA and CaCl₂treatment.Subcellular localization results showed that RtCML42 was localized in the endoplasmic reticulum and RtCDPK16 was localized in the cell membrane.Isothermal titration experiments prove that RtCML42 can play a role in binding Ca²⁺through the EF-Hand region.3.Construction of pPZP221-RtCML42 and pPZP221-RtCDPK16 eukaryotic expression vectors,and transform to Arabidopsis thaliana,wild type and T3generation transgenic Arabidopsis were subjected to drought,salt and ABA stress,and root length,lateral root number,fresh weight and chlorophyll were counted under stress Measured physiological indicators,shows that overexpression of RtCML42 and RtCDPK16 genes improves plant resistance.4.Detection of antioxidant enzyme activity,active oxygen,osmotic adjustment substance and ion content in transgenic and wild-type Arabidopsis under different stress treatments.The content was significantly higher than that of wild type,and the content of H₂O₂,O^2-,MDA was significantly lower than that of wild type,indicating that overexpression of RtCML42 and RtCDPK16 genes can improve the antioxidant capacity of transgenic Arabidopsis thaliana,reduce the degree of membrane damage,and thus improve the transgenic plants,thereby improving the growth of transgenic plants under drought,salt and ABA stress.In addition,the application of a certain concentration of CaCl₂ is further conducive to improving the antioxidant enzyme activity of transgenic Arabidopsis,reducing the accumulation of ROS,and improving its resistance to stress.5.The transgenic and wild-type Arabidopsis thaliana were tested for Na⁺,K⁺,and Ca²⁺.The results showed that compared with wild-type strains,the transgenic Arabidopsis thaliana K⁺and Ca²⁺contents increased significantly,the Na⁺content and Na⁺/K⁺decreased significantly,indicating that the transgenic lines could maintain the low Na⁺/K⁺by regulating the Na⁺,K⁺,Ca²⁺ion content in Arabidopsis thaliana,thus regulating the homeostasis balance of transgenic Arabidopsis.In addition,the application of a certain concentration of CaCl₂ is further conducive to increasing the Na⁺content and Ca²⁺/Na⁺in transgenic Arabidopsis and improving its resistance to adversity stress.6.qRT-PCR analyse show,the expression of genes RtCML42 and RtCDPK16 in Arabidopsis,compare to wild type,the results showed that the expression of antioxidant enzyme genes AtSOD1,AtPOD1,AtAPX1,AtCAT1,ion transport related genes AtSOS1,AtNHXI,AtKUP8,ABA synthesis related genes AtNCED3,and other stress-related genes AtRD29A,AtABF4,AtRD22 were significantly induced,indicating that overexpression of RtCML42 and RtCDPK16genes,it is possible to influence the ABA signaling pathway by inducing the expression of stress-related genes in transgenic plants,regulating the oxidative,osmotic and ion balance of plants under abiotic stress,and improve plant tolerance.