Cloning And Functional Analysis Of Suaeda Salsa SsBR6ox1

BR(Brassinosteroids,BRs or BR)plays an important role in plant salt resistance as the sixth group of widely distributed phytosterol hormone.Suaeda salsa L.is a typical euhalophyte with high salt tolerance.The mechanisms involved in the response of BR to salt stress in halophytes are not yet clear.In this experiment,we cloned the BR biosynthetic enzyme gene SsBR6ox1 from S.salsa and preliminarily verified its function.The results are as follows:1.Physiological mechanism on the role of endogenous BR in salt tolerance of S.salsaS.salsa seedlings from inland saline soils were treated with 500 mM NaCl,and the leaves were sprayed with Brassinazole(Brz,BR biosynthetic specific inhibitor).The results showed that after inhibiting BR biosynthesis by Brz,the shoot biomass,chlorophyll content,SOD activity,endogenous BR content and relative expression of Ss NHX1 were lower than the control(500 mM NaCl without Brz treatment),but the Na~+concentration was higher than the control.The expression of Ss SOS1 tended to be up-regulated in short-term treatment with Brz,but it was down-regulated compared to the control after 3 days.Compared with 0 mM NaCl,BR content in the roots and leaves of S.salsa treated with 500 mM NaCl was significantly increased,and the expression of SsBR6ox1 was up-regulated.The above results suggest that endogenous BR may be involved in salt tolerance of S.salsa through maintaining Na~+ homeostasis via regulating the expression of Ss SOS1 and Ss NHX1.2.Cloning,protein bioinformatics analysis and subcellular localization of SsBR6ox1 geneThe CDS sequence of SsBR6ox1 gene was cloned with a length of 1401 bp according to the sequence of the third generation of S.salsa transcriptome.The pCAMBIA1300-35S-sGFP-SsBR6ox1 expression vector was constructed.SsBR6ox1 gene and the coding amino acid sequence were analyzed on bioinformatics prediction.Ex PASy website showed that the SsBR6ox1 gene encodes 466 amino acids and the SsBR6ox1 protein is hydrophilic;Signal P-4.1 analysis showed that SsBR6ox1 has no signal peptide;TMHMM analysis showed that there is a transmembrane helical region at its tip;SsBR6ox1 has a homology with Lec CYP85A1(75.43%)in Solanum lycopersicum and with CYP85A1(68.52%)in Arabidopsis thaliana,indicating that BR6ox1 is strongly conserved.The preliminary results of SsBR6ox1 subcellular localization showed a fluorescence distribution around the nucleus,preliminar ily indicating that SsBR6ox1 may be localized on the endoplasmic reticulum membrane.3.Screening of Arabidopsis cyp85a1 mutant and SsBR6ox1 transgenic Arabidopsis overexpression line and preliminarily verifying their functionThe Arabidopsis cyp85a1 mutant SALK?101866c was identified by tri-primer method.In addition,SsBR6ox1 overexpression line of Arabidopsis was obtained and screened.Seeds of wild type Arabidopsis(WT),cyp85a1 and SsBR6ox1overexpressed line were sown in 1/2 MS medium with 0 mM and 100 mM NaCl.The results showed that the root length of the cyp85a1 mutant was significantly lower than that of the WT and SsBR6ox1 overexpression line at 0 mM and 100 mM NaCl treatments,while the value in SsBR6ox1 overexpression line was not significantly different from the WT.These preliminary results suggest that SsBR6ox1 may be involved in plant salt tolerance during seedling stages.