Cloning And Functional Analysis Of CIPK Gene From Medicago

Alfalfa?Medicago sativa.?is a large-scale pasture planted in China.It has the advantages of high yield,good quality and good adaptability.Ca²⁺is a major regulator of plant stress and the CBL/CIPK signaling system plays a major role in Ca²⁺regulation.Therefore,this study used molecular biology techniques to study the Medicago truncatula CIPK gene.In this study,five CIPK gene function deletion mutants?cipk3,cipk5,cipk11,cipk18,cipk21?existing in the laboratory were screened,and the highly sensitive phenotype CIPK3 gene was listed as the main research gene.There are high homology?99%?between alfalfa and Medicago truncatula,and Medicago truncatula is a model plant.This study mainly studied the CIPK3 gene of Medicago truncatula R108.The CIPK3 gene sequence was obtained by homologous sequence alignment method,and the CIPK3 gene was subjected to proteomics and functional analysis by bioinformatics techniques.Based on this,The expression level of CIPK3 gene when under different stresses?high salt stress,drought stress and ABA stress?was analyzed by real-time fluorescence quantitative?qRT-PCR?technique;the functional overexpression vector 2×35 SpCAMBIA1300-CIPK3 of Medicago truncatula R108 CIPK3 gene was constructed by Agrobacterium-mediated method.Transformation of Arabidopsis thaliana successfully obtained plants overexpressing the CIPK3 gene,followed by transgenic Arabidopsis for preliminary functional studies.The main findings are as follows:1.The full-length cDNA of Medicago truncatula CIPK3 gene is 1293 bp,encoding 430amino acid residues.The signal peptide prediction results show that the CIPK3 gene encodes a protein that does not contain a signal peptide.There are two transmembrane domains,mainly located in the cytoplasm,which are presumed to be non-secretory proteins that function in the cytoplasm,and with chickpeas,soybeans,and hundred.Leguminous plants such as veins and cowpeas are closely related,but have a close relationship with Arabidopsis,apple,corn,wheat,rice and other plants.2.The CIPK3 gene is expressed in a certain amount in the roots and leaves of the plants,and the relative expression in the leaves is 3.84 times in the roots.When subjected to high salt stress,drought stress and ABA stress,the expression level of CIPK3 gene in each part of the plant showed a certain amount of up-regulation,and the expression level will change according to the different treatment time.When subjected to ABA stress,the relative expression level of CIPK3 gene increased the most,followed by high salt stress and the smallest change under drought stress.The T3 plants of transgenic CIPK3 gene Arabidopsis thaliana were screened and subjected to high salt stress treatment.The wild type was used as a control to observe the phenotype,and the function of CIPK3 gene under stress environment was verified.The results showed that the CIPK3 gene had a positive effect on seed germination,and there was a significant difference in the root length of the plant.It was presumed that it maintained the normal growth of the plant by maintaining the normal ion balance in the plant.In summary,this study finally verified that Medicago truncatula CIPK3 can significantly improve the plant's stress resistance when the plants are subjected to abiotic stress,which lays a foundation for the later study of the highly resistant alfalfa.