| Chloride channels(CLCs)are an important class of anion channel proteins that are widely present in prokaryotic and eukaryotic cells.In terms of plants,they are considered to be associated with cell swelling pressure,osmoregulation,ionic homeostasis,and cellularity,internal pH,stomatal movement,nutrient transport and other functions.Our group has systematically conducted bioinformatics analysis of the CLC homologues and proteins encoded by Arabidopsis thaliana,Glycine max(Linn.)Merr.,rice,and other plant species currently reported,and used related bioinformatics software to synthesize soybean CLCs.Systematic prediction and analysis of the source gene family revealed that seven other homologous gene sequences of the CLC,other than the GmCLCl gene,were sequentially named GmCLC-b1,-b2,-c1,-c2,-d1,-d2,-g,respectively.Our group has completed the gene cloning and sequencing verification and comparison of the above genes,and studied the expression of homologous genes of soybean CLC under salt stress,and found that the transgenic GmCLC-bl,GmCLC-b2,GmCLC-c2 gene on Arabidopsis mutant have higher salt tolerance.In this study,GmCLC-bl and GmCLC-c2 were selected as the research subjects,based on the site-directed mutagenesis(SDM)technique and yeast chloride channel deletion mutant Agefl to study the function of GmCLC-bl and GmCLC-c2 genes.In this study,the soybean GmCLC-bl and GmCLC-c2 genes were constructed into the yeast expression vector pYES2,three highly conserved regions were identified:(?)GxGIPE(?)GKxGPLVH and(?)PVGGVLFALEx,the key amino acids x and several amino acids in the non-conserved region were mutated to other amino acids.The plasmids that were successfully sequenced were extracted and then transformed into yeast chloride channel deletion mutants ?gef1.The main findings are as follows:Under salt stress,G166E,P167L in the conserved region ? of GmCLC-b1 protein,and E210A in conserved region ? could not restore the yeast chloride channel deletion mutant?gef1,and the T437I mutant in the non-conserved region could not resumed growth that the chloride channel deletion mutant ?gef1.The E277G in the conserved region ? and the D601H/P605S mutant in the non-conserved region were able to restore the growth of the yeast chloride channel deletion mutant Agefl to some extent.According to reports in the literature,if the conserved region ? x is P(Proline),NO3-is preferentially transported,and if it is S(Serine),then Cl-is preferentially transported,then the P(Pro)mutate to L(Leu)revealed that P167L could not restore the yeast chloride channel deletion mutant Agefl under salt stress,and the conserved region ? P(Pro)mutate S(Ser),it was found that P167S could restore the growth of the yeast chloride-deficient mutant Agefl under salt stress,and P167L and P167S were inhibited under KNO3 stress.By determining the content of yeast Cl-,the wild-type BY4741,?gef1/GmCLC-b1,conserved regions G166E,P167L,P167S,E210A,E277G and T437I D601H/P605S of the non-conserved region were found that there was no significant change.The GmCLC-b1 protein is expressed as anion transporter activity.Although no Cl" transporting property was observed in this yeast assay,this assay can initially speculate that GmCLC-b1 protein preferentially transports NO3-.Under salt stress,the four mutants of S184F,S184P,E227V,and E294G in the conserved region of GmCLC-c2 protein could not restore the growth of yeast chloride channel deletion mutant ?gef1.Non-conserved C638F,A746T and I71V/C165Y mutants all recovered the growth of yeast chloride channel deletion mutant ?gef1 to some extent.By measuring the Cl-content of yeast,the Cl-content of the S184F,S184P,E227V,and E294G mutants in the conserved region was significantly lower than that of the wild-type BY4741.The x of conserved region I of GmCLC-c2 protein was S(Ser),and the inhibition of S184P was lower than that of S184F under KNO3 treatment.The GmCLC-c2 protein showed anion cotransporter activity,and it was presumed that the protein preferentially transported Cl-. |
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