Functional Analysis Of SbCIPK17、SbCIPK23 And SbCBL1 In Sweet Sorghum(sorghum Bicolor L.) Under Aluminum Stress

In recent years,the issue of soil acidification has aroused global attention.Under acidic soil conditions,Al is released from the solid phase soil and enters the soil solution.When the p H is less than 5.0,micromolar Al(mainly Al³⁺)can quickly cause plant root growth to be blocked,limiting the roots’impact on water and minerals.The absorption of nutrients,which in turn leads to reduced crop yields.Al stress triggers changes in the intracellular Ca²⁺concentration.CBL receives this change and interacts with CIPKs to form a CBL-CIPK signaling system.It participates in the Al tolerance regulation mechanism of plants by regulating the phosphorylation of downstream genes or the expression of Al tolerance genes.Under Al treatment,Arabidopsis Atcbl1 mutant root elongation was significantly inhibited,revealing the potential role of At CBL1 in Arabidopsis resistance to Al stress.The expression of At CIPK17 was significantly down-regulated in Atcbl1mutants,and At CIPK17 could interact with At CBL1.Therefore,it can be inferred that CIPK17,like CBL1,may also participate in the Al stress response of plants.In addition,the transcription level of At CIPK23 gene and the expression level of At CIPK23 protein were up-regulated under Al treatment,revealing that CIPK23may also participate in the Al stress response of plants.However,there is no report on its regulation pathway in sorghum.In this experiment,the Al-tolerant sweet sorghum cultivar ROMA screened by the research group was used as the material,through bioinformatics and expression pattern analysis;subcellular localization analysis;yeast double-hybrid,bimolecular fluorescent complementation（Bi FC）interaction protein analysis and heterogeneous a series of analysis of the phenotype of the source-expressed Arabidopsis thaliana,the function of sweet sorghum SbCIPK17,SbCIPK23 and SbCBL1 were analyzed,and the results of the study are as follows:1、The amino acid sequences of At CIPK17,At CIPK23 and At CBL1 were analyzed by BLAST to obtain homologous proteins of SbCIPK17,SbCIPK23 and SbCBL1.Amino acid sequence analysis showed that SbCIPK17 and SbCIPK23 had conserved activation loop,NAF and PPI domains,and SbCBL1 had conserved EF（Elongation factor-hand）domains and PFPF motifs.Phylogenetic analysis showed that SbCIPK17 and SbCIPK23 had high homology and close relationship with At CIPK17 and At CIPK23,respectively,and SbCBL1 had close relationship with At CBL1.Gene expression pattern analysis showed that the expression of SbCIPK17,SbCIPK23 and SbCBL1 at the transcriptional level were all induced by Al.The relative expression of the underground part is higher than that of the aboveground part.The expression levels of the three genes were the highest at 1-3cm in the root tip,and the expression levels at 0-1cm in the root tip were the most induced by Al.2.The results of subcellular localization test prove that SbCIPK17 and SbCIPK23 proteins are localized in the whole cell.The SbCBL1 protein is located on the cell membrane.3.Yeast two-hybrid nutrient deficiency culture analysis showed that SbCIPK17and SbCIPK23 can interact with SbCBL1,SbCBL2,SbCBL3,and Sb PP2C9.Bimolecular fluorescence complementary interaction experiments further show that SbCIPK17 and SbCIPK23 can interact with SbCBL1 on plant cell membranes.4.Phenotypic analysis of SbCIPK23 overexpressing Arabidopsis strains under Al stress showed that SbCIPK23 and SbCBL1 improved the Al tolerance of Arabidopsis wild-type Col-4.