Functional Analysis Of B HLH85 In Sweet Sorghum Under Salt Stress

In the natural environment,the growth and development of plants is affected by various biotic and abiotic stresses.In order to adapt to the environment,plants can resist external damage through molecular and physiological response mechanisms.The specific expression pathway of the gene is that transcription factors(TFs)interact with cis-acting elements to specifically express related genes in response to environmental stress to maintain the normal life activities of the plants.In this study,the Sbb HLH85 gene was isolated from sweet sorghum,the bioinformatics information of the gene was analyzed,and the expression vector was constructed for subcellular localization analysis.We obtained over-expressed lines of this gene and the homozygous strain of Arabidopsis bhlh85 mutant.The physiological and molecular experiments analysis of wild-type,mutant strains and over-expressed strains were used to confirm the function of Sbb HLH85 gene under salt stress.The main experiments and conclusions are as follows:1.The Sbb HLH85 gene was cloned and the bioinformatic analysis was performed.The sorghum b HLH85 gene has a coding sequence of 951 bp and encodes316 amino acids.It has a typical HLH conserved domain and belongs to the b HLH family.The protein encoded by Sbb HLH85 is an unstable protein,has no transmembrane structure,and has no signal peptide.It is a hydrophilic protein secreted outside the cell.It was found that Sbb HLH85 has the highest homology and the closest relationship with Oryza sativa Japonica Group by comparing the target protein with the homologous protein sequences in other species.2.The cloning vector and expression vector of Sbb HLH85 gene were constructed and transformed into Agrobacterium cells.The gene was expressed in the nucleus by transient transformation of tobacco.3.Overexpressing plants was obtained by transformation of wild-type Arabidopsis thaliana,and then using antibiotic screening and molecular level identification to obtain overexpressing homozygous lines.Overexpression lines OX4 and OX13 with the highest expression were selected by real-time PCR.The bhlh85 mutant seed was purchased from the Tair website and identified by the two-primer method,and the mutant homozygous strain was obtained.The gene expression was identified by real-time PCR,and rsl2-1 and rsl2-3 with lower expression levels were selected for the next experiment.4.The salt-tolerant inbred line M81-E and wild-type Arabidopsis thaliana were treated with 0,50,100,150 and 200 m M Na Cl,and the expression levels of the gene were analyzed in two species.Under salt stress,the expression level of this gene decreased in sweet sorghum,and it was the lowest under 100 m M Na Cl treatment.The expression level of this gene in the roots and leaves of wild-type Arabidopsis thaliana decreased,and decreased with the increase of salt concentration.5.Wild type Arabidopsis thaliana(WT),overexpressing lines(OX4,OX13)and mutant lines(rsl2-1,rsl2-3)were treated with 0,100,150 m M Na Cl,and their physiological indicators of germination were measured.Through germination experiments,we found that the germination rate and root length of mutant lines were better than wild type and overexpressed lines.By measuring the germination rate and root length of each strain under different stresses,it was found that the germination stress was mainly affected by osmotic stress under salt stress.6.Wild type Arabidopsis thaliana(WT),overexpressing strain(OX4,OX13)and mutant strain(rsl2-1,rsl2-3)were treated with 0,100 m M Na Cl,and their physiological indexes were determined.In terms of biomass,mutant strains were higher than WT,and over-expressed lines were lower than WT under salt stress.By performing DAB and NBT staining experiments on each strain,we found that the accumulation of reactive oxygen species was greater in the overexpressing lines,while the accumulation was less in the mutants.From the determination of the MDA content of each strain,it was showed that the malondialdehyde content in the mutant was less than that of the wild type Arabidopsis thaliana and the overexpression strain.In addition,the expression of salt stress-related genes of ionic stress(SOS1,NHX1,HKT1,CLCC),osmotic stress(P5CS1),and oxidative stress(APX,SOD)was analyzed.The expression level of the mutant strain was higher than that of WT and the over-expressed strains.In summary,we can speculate that Sbb HLH85 exhibits a negative regulatory effect under salt stress conditions.7.Wild-type Arabidopsis thaliana(WT),overexpressing Arabidopsis thaliana(OX4,OX13),mutant strains(rsl2-1,rsl2-3),mutant RSL4 that functionally redundant gene of RSL2(rsl4),and the double mutant(rsl2rsl4)was observed during the germination period.We found that the number of root hairs of the overexpressing lines was the highest,and the length of the root hairs was longer than that of the wild type and single mutant lines,and no root hairs appeared in the double mutants.Therefore,we speculate that the root hair growth is closely related to the salt tolerance of plants.Under salt stress conditions,due to the ions absorbed that increase in the number and length of root hairs from the environment increase,resulting in more serious toxicity,so that the salt tolerance is reduced.In summary,the sweet sorghum b HLH85 gene plays a negative regulatory role in the salt tolerance process of plants.