Genome-wide Identification Of BvCBL Genes In Sugar Beet And Resistance Study Of BvSOS1 And BvSOS3

Such as salinity,drought,extreme temperature,K⁺deficiency and insect pests are all kinds of stress encountered in the process of plant growth and development,which are important environmental factors that seriously limit crop growth and yield worldwide.Plants can’t move away from these adversities;they must passively accept them.Therefore,plants have evolved complex signal transduction mechanism and interactive regulatory network during the long-term evolution process,sensing and transmitting signals of external changes,to make timely stress response to adversity.SOS pathway is one of the important ways to respond to salt stress.Three important members in the SOS pathway（SOS1,SOS2,and SOS3）interact with each other to initiate a phosphorylation cascade and expel excessive Na⁺into the external environment,thereby reducing the toxicity of high salt to cells.Therefore,SOS pathway plays an important role in maintaining the balance of K⁺/Na⁺in plant cells and reducing the accumulation of Na⁺in cells and even in plants.Therefore,it is necessary to clone the key genes of sugar beet SOS pathway and study its function.In this paper,the BvCBLs gene family was excavated and identified at the whole genome level in sugar beet（Beta vulgaris L.）.qRT-PCR was used to analyze its expression patterns under salt and drought stress.In addition,the full-length cDNA of BvCBL1（BvSOS3）and plasma membrane Na⁺/H⁺antiporter BvSOS1 were cloned,and pEarley Gate 100-BvSOS1/3 overexpression vector was constructed,which was genetically transformed into Arabidopsis for verification of BvSOS1/3 salt tolerance.The results obtained are as follows:（1）Seven BvCBLs genes were identified in genome of sugar beet.Phylogenetically,BvCBLs can be divided into four groups（GroupⅠ,Ⅱ,Ⅲ,andⅣ）.All BvCBLs contain conserved EF-Hand motifs,while the N-terminus of BvCBL1,-2 and-5 contain myridamoylated sites,and the N-terminus of BvCBL4,-6 and-6 contain a transmembrane domain.The predicted BvCBLs interact with multiple CIPKs.In addition,qRT-PCR results showed that under 50 mmol/L NaCl treatment,the expression pattern of BvCBLs in roots and shoots was increased significantly,while under sorbitol treatment,the expression pattern of BvCBLs in roots was significantly induced.These results suggest that BvCBLs may play an important role in sugar beet response to salt and drought stress.（2）BvSOS3（BvCBL1）and the plasma membrane Na⁺/H⁺antitransport gene BvSOS1 were cloned from sugar beet by designing primers based on the sequences of Beta vulgaris Resource.The ORF of BvSOS1 was 3489 bp,encoding 1126 amino acids,and the OFR of BvSOS3 was 660 bp,encoding 219 aa,both of which were hydrophilic proteins.The target fragment of BvSOS1/3 was cloned into overexpression vector pEarley Gate 100,and two pEarley Gate 100-BvSOS1/3 overexpression vectors were constructed and identified by PCR,enzyme digestion and sequencing,and then transferred into agrobacterium GV3101.Then,the overexpression vector containing BvSOS1/3 target fragment was transformed into Arabidopsis thaliana plants by flower infection method,T₁ generation seeds were collected,and T₃ generation homozygous plants were obtained by Basta resistance screening for functional verification.（3）BvSOS1/3 transgenic plants screened by Basta were confirmed by genomicDNA PCR,sequencing and qRT-PCR to transfer BvSOS1/3 gene into Arabidopsis thaliana,and BvSOS1/3 transgenic positive lines were obtained.The expression of BvSOS1/3 in transgenic positive plants was 4000-15000 times higher than WT plants.The seeds of T₃ homozygous plants were treated with 150 mmol/L NaCl and 0.5μmol/L ABA,and the germination rate of seeds of BvSOS1/3 overexpressed line was significantly higher than that of the WT.BvSOS1/3 transgenic positive plants were treated with 75 and 150 mmol/L NaCl,and their physiological parameters such as root length,fresh weight,proline and chlorophyll content and the expression levels of related stress genes（AtSOD1,AtCAT1,AtPOD1,and AtP5CS1）were significantly increased.These results indicated that the overexpression of BvSOS1/3 gene may enhance the osmotic regulation and scavenging capacity of reactive oxygen species in Arabidopsis thaliana,and thus enhance the tolerance of plants to salt.In conclusion,based on the identification of sugar beet CBL family members and the expression pattern analysis under stress,this study combined with the overexpression of BvSOS1 and BvSOS3（BvCBL1）genes in Arabidopsis to verify the mechanism of sugar beet SOS pathway in response to salt stress.In order to provide genetic resources and theoretical basis for improving crop stress resistance.