| Saline-alkali stress is one of the abiotic stresses that affect growth and development.Sulfur transporter is a carrier protein that absorbs and transports sulfate and play an important role in plant growth resistance to abiotic stress.In this study,the MsSULTR gene of Medicago sativa was cloned and transformed into Arabidopsis thaliana.The function of MsSULTR gene was verified by analyzing the phenotypic and physiological characteristics of transgenic Arabidopsis thaliana under saline-alkali stress.The main research results are as follows:1.Cloning and bioinformatics analysis of MsSULTR geneThe MsSULTR gene of alfalfa was cloned by RT-PCR.The gene full length is 1677bp,and coding 558 amino acids.MsSULTR protein has a transmembrane domain and belongs to hydrophobic protein.In its secondary structure,αhelix accounts for 48.92%,βcorner accounts for 4.30%,irregular crimping accounts for 32.08%,and extension chain accounts for 14.70%.Evolutionary tree analysis showed that Medicago sativa MsSULTR was the closest relative to Medicago truncatula Mt SULTR,and sequence alignment showed that there existed nine differential sites between MsSULTR and Mt SULTR,and five amino acid differential sites in the conserved domain.2.Tissue-specific expression and abiotic stress expression of MsSULTR geneMsSULTR gene was expressed in the root,stem,leaf,flower and pod of alfalfa,with the highest expression level in pod and the lowest expression level in root.Under salt treatment,MsSULTR gene showed an upward trend at 3 h,12 h and 24 h,but there was no significant change at other time points.Under saline-alkali treatment,the expression of MsSULTR showed an upward trend,and reached its peak at 12 h.Under low temperature treatment,MsSULTR gene increased at 1 h and 24 h,but decreased significantly at other time points.Under drought treatment,the expression of MsSULTR was up-regulated except for 1 h and 12 h,and the expression was the highest at 24 h.3.Construction of p Early Gate-MsSULTR expression vector and genetic transformation of Arabidopsis thaliana.The cloned MsSULTR gene was linked to the expression vector p Early Gate-1×Flag to form the p Early Gate-MsSULTR-1×Flag plant expression vector.The cloned MsSULTR gene was ligated with the intermediate vector p GWC to obtain the plant intermediate vector p GWC-MsSULTR,and then connected with the expression vector p Early Gate-1×Flag to construct the p Early Gate-MsSULTR-1×Flag plant expression vector.The MsSULTR gene was introduced into Arabidopsis thaliana,combined with PPT resistance screening and PCR identification,10 transgenic Arabidopsis plants with MsSULTR gene were obtained.The results of q RT-PCR detection showed that the target genes were well expressed in strains 6 and 8,which could be used for follow-up gene function analysis.Arabidopsis mutant s268 was homozygous by triple primer method.The results showed that Arabidopsis mutant s268 was a homozygous mutant.4.Functional analysis of MsSULTR gene under sulfur deficiency treatmentIn this study,Arabidopsis thaliana mutants and reverting mutants were analyzed under sulfur deficiency treatment.the results showed that the root length and fresh weight of restoring mutants were roughly the same as those of wild-type Arabidopsis thaliana in sulfur-deficient treatment.The root length and fresh weight of the mutant plants were inhibited,in addition,the root length and fresh weight of the mutants were more lacking than those of the wild-type plants,indicating that the restoring mutants could restore the tolerance of Arabidopsis to sulfur deficiency stress.Through the determination of GSH content,it was found that the GSH content of Arabidopsis thaliana mutant s268 was less under sulfur deficiency treatment,while the content of Arabidopsis thaliana reverting mutant was roughly the same as that of wild-type Arabidopsis thaliana.At the same time,the expression of three key enzymes genes ATPS,Si R and OASTL during sulfur assimilation under sulfur deficiency treatment further indicated that sulfate transporters play an important role in the process of sulfur assimilation.5.Analysis of saline-alkali tolerance of transgenic Arabidopsis thaliana with MsSULTR GeneTransgenic Arabidopsis thaliana seedlings with MsSULTR gene were treated with saline-alkali stress.The results showed that the root length and chlorophyll content of Arabidopsis seedlings treated by saline-alkali stress were significantly higher than those of wild type,however,the Arabidopsis s268 mutant had lower tolerance to saline-alkaline,chlorophyll content decreased obviously,while root growth was severely inhibited.Under 150 m M Na HCO3treatment,the transgenic MsSULTR gene,wild-type and Arabidopsis mutants also exhibited similar phenotypes.The contents of sulfate and glutathione in transgenic MsSULTR Arabidopsis were higher than those in wild-type and mutant s268.The contents of proline,soluble protein and soluble sugar in MsSULTR transgenic Arabidopsis were significantly higher than those in wild type and s268,MDA,O2-and H2O2content were lower than those of wild type and s268.Meanwhile,DAB,NBT staining of Arabidopsis leaves after saline-alkali stress showed that MsSULTR transgenic Arabidopsis was less heavily stained degree than wild type and the mutant was more heavily stained degree than wild type.The results showed that transgenic Arabidopsis thaliana with MsSULTR gene showed strong saline-alkali resistance,while the Arabidopsis mutant was least saline-alkali tolerant. |
