Genome-Wide Analysis Of Glutathione S-Transferase（CqGSTs） And Study On Their Salt Tolerance Function In Root System In Quinoa （Chenopodium Quinoa Willd.）

Since the industrial Revolution,as the global economy has boomed and the agricultural population has expanded.Excessive use of chemical fertilizer and unreasonable farming methods make soil salinization more and more serious.Soil salinization has become one of the important factors affecting crop yield and quality and restricting agricultural development.Chenopodium quinoa,it has unique nutritional value and strong stress resistance and adaptability,under the background of soil salinization,quinoa has been widely studied as a halophyte model.With the release of high-quality genome of quinoa,more and more salt-tolerant genes of quinoa have been cloned gradually.Glutathione S-transferases（GSTs）are a kind of multifunctional enzymes widely existing in animals,plants and microorganisms.They are widely involved in the detoxification function of plants and play an important role in resisting biological and abiotic stresses.It has been proved that GST gene plays an important role in resistance to salt stress in rice,tomato,soybean and other plants.At present,whether the GST gene of quinoa also plays an important role in salt stress has not been known.Therefore,this study carries out three aspects of work.Firstly,the whole genome of quinoa was identified by bioinformatics and the genetic relationship,physicochemical properties,gene structure,chromosome location,evolutionary relationship and expression pattern of GST gene were analyzed.Secondly,The root transformation system induced by agrobacterium tumefaciens was established and optimized to solve the problem of difficult transformation of quinoa.Finally,The function of GST gene in selected quinoa were identified by the established root transformation system.The main results are as follows.（1）Bioinformatics and expression analysis of GST gene in quinoaIn this study,114 CqGST genes were identified from the whole genome of quinoa by bioinformatics methods.The phylogenetic tree showed that 114 CqGST genes were divided into seven subgroups:GSTU（68 members）,GSTF（23 members）,GSTL（6members）,GSTZ（6 members）,GSTT（5 members）,DHAR（4 members）and TCHQD（2members）.Gene structure and Motif analysis showed high similarity among members of each subgroup.Phylogenetic analysis of these genes suggested that tandem and fragment replication events played a key role in the expansion of the CqGSTs gene family,and the CqGST genes may have undergone strong purification selection during the evolution process.Analysis of salt-treated transcriptome from the roots of salt-tolerant and salt-sensitive quinoa cultivars showed that Salt treatment induced changes in the expression levels of CqGSTs genes,and eight CqGST genes（CqDHAR2、CqDHAR3、CqGSTU22、CqGSTU44、CqGSTU60、CqGSTU63、CqGSTU67、CqGSTU68）were steadily up-regulated in both cultivars.RT-qPCR results showed that these selected CqGST genes were not only induced by salt stress,but also by drought stress.（2）Construction of binary expression vector and optimization of quinoa root transformation systemBecause the transformation system of quinoa is not mature,it is still difficult to study the function of quinoa.Therefore,this experiment established and optimized an efficient and rapid transformation system of quinoa root induced by Agrobacterium（Ar.A4）.The p K7WGF2::hp Cas9 vector was successfully modified to verify the function of quinoa GST gene in quinoa root system.The results showed that the conversion efficiency of hypocotyl infected quinoa reached the highest when the concentration of OD600 was 0.6～1.0 after 25～30 days of quinoa growth.In addition,the conversion efficiency was related to quinoa varieties,and Dave had the highest conversion efficiency,reaching 80%.Eight genes were selected to respond to salt stress and constructed into the modified vector p K7WGF2::RFP for further functional study.（3）Root expression analysis and functional identification of GST gene in quinoaThe roots of positive transgenic quinoa were treated with 400 mmol/L NaCl for 48 h and collected for further analysis.RT-qPCR results showed that GST genes expression in positive transgenic quinoa roots（TRs）was significantly increased by 2～8 times compared with negative transgenic quinoa roots（ARs）.The results further verified the success of quinoa root transgenic and proved that quinoa GST gene played an important role under salt stress.DAB staining and H₂O₂content determination showed that TRs significantly reduced the accumulation of H₂O₂compared with ARs.Compared with ARs,GSH content in roots of CqDHAR2,CqGSTU22,CqGSTU44,CqGSTU63 and CqGSTU68 were significantly increased.In conclusion,the transgenic roots of CqDHAR2,CqGSTU22,CqGSTU44,CqGSTU63 and CqGSTU68 balanced the endogenous hydrogen peroxide induced by salt stress,and alleviated the damage caused by hydrogen peroxide to the root cells of quinoa.