Molecular Mechanism For ERF96 Gene In Regulating Selenium Tolerance In Arabidopsis Thaliana

It's believed that ethylene-related genes regulate a variety of stress responses.In the study of plant resistance to selenium stress,it was found that the expression level of ethylene response factor(ERF)subfamily ERF96 gene in Arabidopsis thaliana was significantly increased under selenium induction.However,the function of ERF96 gene under selenium stress signal and the molecular mechanism for regulating selenium stress response are still unknown,so this problem was further discussed and the research results are as follows:1.Construction of the ERF96 overexpression vector and generation of ERF96-overexpression plant.Specific primers were used to amplify fragments of ERF96 coding region by PCR and connected them to the Entry vector.The gene fragment was then transferred to the vector of p OVER-Kan with 35 S promoter by LR reaction,and the positive strain was identified after the vector of p OVER-ERF96-Kan was transferred to Agrobacterium,which has finally transferred to the wild type of plant,and the positive ERF96 overexpressed homozygous plants OE-1,OE-2 and OE-3 were screened and identified.2.Construction of clustered regulatory interspersed short palindromic repeatassociated endonuclease(CRISPR)vector and generation of erf96 mutant lines.Three CRISPR target sites g RNA1/g RNA2/g RNA3 were selected from ERF96 DNA,the U6 promoter fragment was cloned and fused with three g RNA-scaffold fragments respectively.The obtained 96-G1/G2/G3 cassettes were fused with p ENTRY vector by digestion and ligation,then transferred to the target vector to obtain the CRISPR vector of ERF96.The final vector was transferred to Agrobacterium GV3101 for preservation.The CRISPR vector was transferred into Arabidopsis thaliana Col-0 through floral dipping,and the ERF96 DNA-related PCR products of T1 and T2 strains were digested by T7 EI enzyme for screening.We obtained homozygous seeds of the ERF96-knockout alleles erf96-2 and erf96-3,in which 92 bp and 89 bp deletion in two different sites of the ERF96 gene,respectively.3.The selenium stress phenotypes of seeds of transgenic plants and mutant lines were analyzed.In the selenite stress condition,the root length and fresh weight of ERF96 overexpression plants were significantly higher than that of wild type.It showed selenium tolerance.However,there was no significant difference between the mutant and the wild-type(WT)line in selenium containing environment,at the same time,under Se treatment,the Se content in erf96-2 and erf96-3 were similar to WT.It is speculated that this may be the result of gene functional redundancy,which is very common in ERFs.4.Overexpression of ERF96 gene affected the absorption and distribution of selenium in Arabidopsis thaliana.Under the stress of sodium selenite,Se accumulation in the shoot part of ERF96 overexpression plants was less than that in the wild type.q RT-PCR analysis of selenite/phosphate transporters revealed that transcriptional levels of PHT1;1 and PHT2;1 in overexpression lines was significantly reduced under selenite stress.These results indicated that ERF96 reduces plant uptake of selenium and its transfer to shoot by decreasing the expression level of PHT1;1 and PHT2;1.The result was helpful to selenium tolerance of the overexpression plants.5.Overexpression of ERF96 gene enhanced the antioxidant activity of Arabidopsis thaliana.Se treatment induced oxidative stress and accumulation of reactive oxygen species.Overexpression of ERF96 under selenium stress enhanced the activity of catalase(CAT)and glutathione peroxidase(GPX)in plants,it also increased the content of glutathione(GSH)content and the gene expression level of GSH1,indicated that ERF96 improved the antioxidant capacity of Arabidopsis thaliana under selenium stress.Moreover,the addition of the GSH synthesis inhibitor buthionine sulfoximine(BSO)into the medium resulted in the loss of OE-1 resistance to selenite stress.These results indicated that the selenium tolerance of ERF96 overexpression plants required GSH.In summary,our study provides genetic evidence that ERF96 regulates Se tolerance in Arabidopsis thaliana through reducing selenium absorption and distribution and emhancing antioxidant activity.