Functional Analysis Of Arabidopsis GSTF11/U20 And MYB28 In The Synthesis Of Aliphatic Glucosinolates

Glucosinolate is a kind of nitrogen and sulfur containing plant secondary metabolites derived from amino acids,mainly found in cruciferous plants.The glucosinolate and its degradation products have a variety of biological activities,especially anti-tumor effects,and thus have attracted wide spread attention.In Arabidopsis,Glutathione S-transferase(GSTs)plays an important role in glutathione metabolism by catalyzing the binding of acid nitrocompounds or nitrile to glutathione.However,the specific GST involved in the synthesis of aliphatic glucosinolates and the biological functions of GST in the synthesis of aliphatic glucosinolines remain to be further studied.The synthesis pathway of glucosinolate metabolism is quite clear.More effort should be put on the regulatory mechanism of glucosinolates metabolism.Previous studies have shown that the key transcription factor MYB28 of glucosinolate synthesis not only directly regulates the expression of the glucosinolate synthesis pathway gene,but also may regulate other metabolic processes.The regulation network of MYB28 has not been clear so far.Whether MYB28 regulates glucosinolates synthesis by mediating other metabolic pathways remains to be investigated.Glutathione is a donor of sulfur in the synthesis of glucosinolates.In view of the above problems,this study analyzed the functions of GSTF11 and GSTU20 in glucosinolide synthesis and the regulatory network of MYB28,a key transcription factor that regulates glucosinolide synthesis.On the one hand,the GSTF11 and GSTU20 genes were edited using the CRISPR/Cas9 system to obtain the single mutant strains of gstfll and gstu20,and then the functions of GSTF11 and GSTU20 in the synthesis of glucosinolate were analyzed.On the other hand,On the one hand,the combination of ChIP-Seq and RNA-Seq was used to identify the binding gene and downstream regulatory gene of MYB28 in the whole Arabidopsis thaliana genome,and the regulatory network of MYB28 transcription factor was analyzed.The main results were as follows:(1)The co-expression network of aliphatic glucosinolates synthesis genes showed that GSTF11 and GSTU20 were correlated with the genes involved in the synthesis of aliphatic glucosinolines in Arabidopsis thaliana.Combined with GUS staining and Real-time quantitative PCR analysis,it was found that GSTF11 and GSTU20 were expressed in the roots,stems,leaves,flowers and silips of arabidopsis thaliana.Among them,GSTF11 was highly expressed in leaves,while GSTU20 was the most expressed in silips.Gene edited using the CRISPR/Cas9 system,get gstf11 and gstu20 gene fragment missing mutants,and found that gstfll glucosinolates and gstu20 significantly reduce,especially aliphatic glucosinolates content and glucosinolates gstfllgstu20 double mutant of aliphatic content significantly lower than the single mutants,showed that gstfll and gstu20 glucosinolates in Arabidopsis aliphatic synthesis,at the same time,preliminary showed that gstfll and glucosinolates gstu20 genes in synthesis function of redundancy.(2)Identification to 745 and 2967 significant differentially expressed genes by RNA-Seq respectively in gstf11 and gstu20 mutant,including glutathione metabolism and glucosinolate metabolism related genes get significant enrichment.Glutathione is an important sulfur donor for glucosinolates synthesis,GSTF11 and GSTU20 may play an important role in the synthesis of glucosinolates by regulating glutathione synthesis.(3)MYB28 with MYC tag at N and C-terminal was successfully cloned,then used to construct 35S::MYB28:MYC transgenic plant with pCAMBIA1305 as destination vector.The transgenic plants were obtained by the method of Agrobacterium-mediated transformation.Western blot and Real-time fluorescent quantitative PCR were performed to determine the expression of MYB28 at protein and mRNA level,also the content of glucosinolate was determined to 35S::4xMYCMYB28 expression plant 7#was selected as the best transgenic strain and used for the subsequent ChIP-Seq analysis.(4)ChIP-Seq results showed that a total of 754 Peaks were enriched,among which 735 MYB28 binding genes were located in the gene region.The MYB28 binding site in the genome is mainly located in the promoter region,and the conservatively bound Motif TGGGC.The enrichment analysis of metabolic pathways showed that MYB28 binding genes were mainly involved in the metabolism of galactose,RNA transport,glutathione metabolism,starch and sucrose metabolism,citric acid cycle,fructose and mannose metabolism,glycolysis and other metabolic pathways.Among MYB28 binding genes,we found that HXK1,PFK7,PGM2,PED345,CSY2,ICDH and cICDH are key genes regulating sugar metabolism,suggesting that MYB28 may mediate sugar metabolism through transcriptional regulation of these genes,and then indirectly regulate glucosinolates synthesis.In addition,we screened two genes related to glucosinolide synthesis,IGMT5 and GSH1,and speculated that MYB28 might directly regulate glucosinolide synthesis by binding to these two genes.(5)1028 significantly differentially expressed genes were identified in myb28 mutant.Differential genes were highly enriched in glucosinolates synthesis,glutathione metabolism,and defense response pathways,which confirmed the important role of MYB28 in glucosinolates synthesis.In summary,GSTF11 and GSTU20 play an important role in glucosinolidin synthesis,and the potential mechanism may be to regulate the anabolism of glucosinolidin by regulating glutathione synthesis and affecting the sulfur donors in the pathway of glucosinolidin synthesis.MYB28 not only directly regulates the expression of genes related to the synthesis pathway of aliphatic glucosinolates,but also may mediate carbohydrate metabolism through transcriptional regulation,thus regulating the synthesis of glucosinolates in Arabidopsis thaliana.