Cloning And Genetic Transformation Of Iron Deficiency-related BHLH Transcription Factors In (Medicago Sativa L.)

Iron is one of the essential trace elements for plant life activities which easily to be deficiency in plants because of the effective iron content in soil is low.The bHLH transcription factor family plays important regulatory roles in plant,and some of bHLH transcription factor are involved in response to iron deficiency in plant.In this study,MsbHLH115 gene was cloned from alfalfa,pBI121-MsbHLH115 plant expression vector was constructed and transferred into wild type tobacco,and the expression mode of MsbHLH115 gene was preliminarily studied.The main research results are as follows:1.Cloning and sequence analysis of MsbHLH115 geneA clone primer was designed based on transcriptomic sequence under iron stress of alfalfa.MsbHLH115 gene was cloned by RT-PCR method.The total length of the gene was 780 bp,encoding 259 amino acids.MsbHLH115 protein contains an HLH domain.BLAST protein analysis showed that the amino acid similarity of MsbHLH115 and MtbHLH115 was 96%.Based on the prediction of MsbHLH115 amino acid sequence and bioinformatics analysis,the tertiary structure model and phylogenetic tree were constructed.2.Construction of MsbHLH115 plant expression vectorPlant expression vector pBI121-MsbHLH115 was constructed,containing Ca MV35 S promoter and kanamycin(Km)resistance.3.Genetic transformation of MsbHLH115 gene into tobaccoThe recombinant plasmid pBI121-MsbHLH115 was transferred into the GV3101 agrobacterium,and then MsbHLH115 was transformed into model plant tobacco by leaf disc method.Two transgenic positive seedlings were identified by PCR.4.Expression of MsbHLH115 geneThe relative expression of MsbHLH115 gene was detected by qRT-PCR in alfalfa under different abiotic stress.It was found that MsbHLH115 gene was strongly induced by high iron and iron deficiency.The expression was strongly induce in root while not in stem or leaf;MsbHLH115 was down-regulated in response to salt stress,up-regulated in response to alkali stress or drought stress;MsbHLH115 was up-regulated in response to ABA,while not in response to methyl jasmonate.