| Plant hormones are important substances that regulate the growth and development of plants by regulating the relative expression of certain genes,including cell development,morphogenesis,stress response,and immune response.As a novel recognized phytohormone,strigolactones are a general term for a class of substances,which have multiple structures and types in plants.Strigolactones play an important role in regulating plant branching,as well as root configuration,leaf senescence and plant stress response.SMXLs genes are homologous genes of rice D53 gene.In Arabidopsis thaliana,SMXLs family contains 8 members,which are divided into three subfamilies.Among them,AtSMXL7 has the highest homology with D53,which encodes a strigolactones repressor protein.In this study,‘Royal Gala’ apple was used as the research object.By homologous sequence alignment,we found the MdSMXL8.2 gene,which was highly homologous with AtSMXL7 in Arabidopsis.The gene structure and chromosomal location,protein structure,cis elements of its promoter were analyzed.Its expression pattern in different organs as well as the response to abiotic stress and other hormones were analyzed.Then,analysis of its effect on plant salt resistance were performed using MdSMXL8.2 overexpressing apple calli and heterologous over expressed Arabidopsis thaliana.Besides,its affection on shoot branching was investigated.The specific work and main research results are as follows:1.Bioinformatics analysis of MdSMXL8.2Analysis of gene structure and chromosomal location showed that MdSMXL8.2 contained three extrons as well as two introns.Its open reading frame was 3243 bp,which encoded 1080 amino acids.MdSMXL8.2 was located on chromosome 07 of apple.Besides,MdSMXL8.2 had an alternative splicing in apple,which was caused by intron retention.Conserved protein domain analysis showed that MdSMXL8.2 had two Clp domains.Phylogenetic tree analysis between different species revealed that MdSMXL8.2 was most homology with Arabidopsis AtSMXL7,and the 3D conformation of MdSMXL8.2 highly matched the 3D conformation of AtSMXL7.The in-silico analysis suggested that the promoter sequence of MdSMXL8.2 contained variety cis-acting elements,which included hormone-responsiveness,stress-responsiveness and plant development regulation related cis elements.2.Expression pattern analysis of MdSMXL8.2qRT-PCR showed that MdSMXL8.2 was mainly expressed in stems and leaves.Hormonal response analysis suggested that ABA induced MdSMXL8.2 expression and MdSMXL8.2 also responded to IAA,SA and MeJA.Stress response analysis indicated that NaCl repressed MdSMXL8.2 expression while PEG6000 and mannitol induced MdSMXL8.2 expression.3.MdSMXL8.2 negatively regulates plant salt resistanceMdSMXL8.2 over expressed apple calli and Arabidopsis was obtained by Agrobacterium tumefaciens-mediated transformation.Further analysis indicated that MdSMXL8.2 over expressed apple calli and Arabidopsis was more sensitive to salt stress compared with the wild type.Transgenic plant materials were significantly worse than the wild type and its cells were more severely damaged under salt stress.Additionally,MdSMXL8.2 over expressed apple calli was more sensitive to osmotic stress.4.MdSMXL8.2 influences plant branchingThe in vitro protein degradation experiment revealed that MdSMXL8.2 protein was not stable enough and could be degraded by rac-GR24,which was a biologically active strigolactone synthetic analog.Statistics on the branches of MdSMXL8.2 overexpressing Arabidopsis showed that rosette leaf branches and main branch secondary side branches increased by 1 on average,which indicated that MdSMXL8.2 influences plant branching. |
