Functional Analysis Of Glutathione Transferase Gene BnGSTU12 Against Sclerotinia Disease In Brassica Napus

Stem rot casued by Sclerotinia sclerotiorum is a major disease in Brasscia napus production,which can cause a decrease in yield and quality of rapeseed.Glutathione S-Transferases（GSTs）are multifunctional proteins encoded by a large gene family.GSTs play the important functions in response to stress,detoxification,stress resistance,signal transduction and so on.GST has glutathione peroxidase activity,which can reduce organic hydroperoxides and prevent plants from oxidative damage under stress.Nowadays,researches about the GST gene families in plants mainly focuses on the resistance to biological and abiotic stresses in rice,poplar,and Arabidopsis thaliana.However,research about the function and mechanism of GST gene in the resistance to S.sclerotiorum in the B.napus has not been reported.In the previous research,we evaluated stem resistance to S.sclerotiorum in 347 B.napus accessions-using the detached stem inoculation with the Illumina 60K SNP chip.Through genome-wide association analysis,we detected 17 SNP loci significantly associated with stem resistance.In addition,RNA-seq was performed 48 hours after inoculation with resistant and susceptible B.napus lines.Combining genome-wide association analysis and RNA-seq,we found that a glutathione s-transferase（GST）gene cluster was upregulated（BnaC06g31020D,BnaC06g31030D,and BnaC06g31040D）;at the same time,the differentially expressed genes before and after inoculation of S.sclerotiorum enriched in the glutathione metabolism process.We used the whole genome resequencing（5x）data of B.napus to perform candidate association analysis of GST candidate genes,and identified BnGSTU12（BnaC06g31030D）as a candidate disease resistance gene.Based on previous studies,this study analyzed the expression patterns of BnGSTU12gene in B.napus and under different stresses through qRT-PCR,and genetically transformed A.thaliana and B.napus.Combining enzyme activity analysis,transient expression of Nicotiana benthamiana was performed to study the function of candidate gene BnGSTU12 in the resistance to S.sclerotiorum in A.thaliana and B.napus.The hormone content and antioxidant enzyme activities before and after inoculation with S.sclerotiorum were measured,and the mechanism of BnGSTU12 gene in disease resistance was analyzed.This study not only was important for the theoretical study of resistance to S.sclerotiorum in rapeseed,but also can provide new strategies for resistance breeding in rapeseed.The main research results are as follows:（1）The tissue expression pattern of BnGSTU12 in Zhongshuang 11 showed that BnGSTU12 was expressed in various tissues of B.napus,with a high expression level in roots,stems,leaves,stamens,and seeds.Staining analysis of A.thaliana with GUS activity showed that Pro BnGSTU12 was also stained on roots,stems,leaves,flowers,consistent with the results in qRT-PCR.In this study,B.napus was inoculated with S.sclerotiorum,and it was found that the expression of BnGSTU12 gene increased 24 hours after inoculation of B.napus leaves with S.sclerotiorum,indicating that S.sclerotiorum treatment can induce BnGSTU12 expression.We analyzed the gene expression of BnGSTU12 after PEG（drought）,Na Cl（salt）,and UV treatment,and found that the expression of BnGSTU12 gene was significantly affected by UV.After treatment with different hormones（ABA,GA3,KT,ETH,SA,Me JA）,BnGSTU12 was induced by ETH,SA,and Me JA.（2）After inducing the expression of the recombinant protein and determining its enzyme activity,we detected a protein band in about 29 k Da in the induced Escherichia coli,which was consistent with the expected protein size;BnGSTU12 has catalytic activity for CDNB.（3）In this study,a plant overexpression vector induced by 35S promoter and a VIGS silencing vector were constructed.After genetic transformation of A.thaliana and B.napus using plant overexpression vectors,it was found that compared with wild-type A.thaliana,the T₃ generation BnGSTU12 overexpression transgenic A.thaliana had a reduced lesion area after identifying the resistance to S.sclerotiorum in the detahed leaves.In addition,the lesion area of the leaves of 12 hpi,24 hpi,and 36 hpi in the overexpressed strains was smaller than WT.In B.napus,the T₂ generation BnGSTU12 overexpression transgenic rapeseed had a smaller lesion area than WT.After genetic transformation of B.napus using the VIGS silencing vector,the results revealed that the T₁ generation transgenic rapeseed had an increased lesion area compared to the ZS11 transformed with the empty vector.Using a plant overexpression vector to instantaneously express N.benthamiana,we found that the lesion area of N.benthamiana leaves containing pL-35S-Ds Red-BnGSTU12 was reduced compared to N.benthamiana leaves transformed with an empty vector.The above results indicated that BnGSTU12 could enhance the resistance of plants to S.sclerotiorum.（4）In this study,we measured the contents of SOD,POD,CAT,GST enzyme activity,H₂O₂,GSH,and GSSG before and after inoculation with S.sclerotiorum.The results showed that SOD,POD,and CAT enzyme activities in overexpressed A.thaliana were higher than those in wild-type plants.The H₂O₂ content increased at 6 h after inoculation and then decreased at 24 h,and H₂O₂ content in overexpressed A.thaliana lines decreased more than WT.The activity of GST enzyme in overexpressed A.thaliana was significantly higher than WT 24 h after inoculation.Using DAB staining to detect the accumulation of H₂O₂ in A.thaliana leaves,we found that there was a large area of red sediment in the leaves of WT plants,and only a small amount of red sediment in overexpressed A.thaliana,indicating that overexpression of BnGSTU12 gene led to less ROS in A.thaliana.We also measured the SOD,POD and CAT enzyme activities in WT and overexpressed B.napus,we found that SOD and CAT enzyme activities were always higher than those in wild type plants,and POD activity was higher than that in wild type plants after inoculation with S.sclerotiorum for 24 hours,with a decrease in H₂O₂ content.With the increase of inoculation time,the content of GSH,GSSG,and GST activity in BnGSTU12 overexpressed rapeseed were higher than those in the control.The above results indicate that overexpression of BnGSTU12 gene increased the activity of antioxidant enzymes in A.thaliana and B.napus,reducing the plants damage caused by reactive oxygen species.（5）In this study,the content of JA and SA was measured before and after inoculation with S.sclerotiorum in overexpressed A.thaliana and B.napus.Compared with WT,the content of JA in overexpressed A.thaliana significantly increased at 18 h after inoculation.The content of SA in overexpressed A.thaliana decreased.In B.napus,the JA content of overexpressed rapeseed increased before and after 12 hours of inoculation compared to WT,while it decreased after 24 hours of inoculation.However,the SA content in the overexpressed plants decreased after inoculation.The results showed that overexpression of BnGSTU12 gene induces JA synthesis and enhances the resistance of A.thaliana and B.napus to S.sclerotiorum.（6）In this study,key genes in the JA and SA synthesis and signaling pathways using qRT-PCR analysis.Compared to wild type A.thaliana,the expression of genes involved in the JA synthesis pathway in BnGSTU12 overexpression A.thaliana,AtLOX2,AtOPR3,and AtAOC1,did not significantly change,while the expression of AtJMT,AtJAR1,AtCOI1,and AtPDF1.2 in JA signaling pathway were upregulated after inoculation with S.sclerotiorum.The expression of AtJAZ1 gene was down regulated.The expression of AtNPR1,a key gene of SA signaling pathway in A.thaliana,was not induced,and the expression of AtPR3 was significantly down regulated.The above results indicate that overexpression of the BnGSTU12 gene activates the JA signaling pathway and increases the resistance of A.thaliana and B.napus to S.sclerotiorum.