Screening Of Toxicodendron Vernicifluum Glutathione S-transferase Gene Family And Cloning And Functional Analysis Of Lambda GST Gene

Lacquer tree(Toxicodendron vernicifluum(Stokes)F.A.Barkl.)is one of the most ancient multi-purpose tree in China.Due to the limitation of climate,water and heat conditions,excessive utilization and insufficient protection,the resources of lacquer trees and raw lacquer in China are continuously decreasing.Glutathione S-transferases(GSTs,EC2.5.1.18),which are widely present in the cytoplasm,are closely related to plant stress resistance,and play an important role in the removal of oxidative damage caused by either biological or abiotic stresses.Therefore,the study of glutathione S-transferase is very important to reveal the stress resistance mechanism of lacquer tree.In this study,the main variety of lacquer tree,"Dahongpao",was used.Based on the lacquer tree transcriptome data,38 GSTs gene sequence are obtained.Among these,the Lambda GST gene was cloned,expressed and its function was studied.The main research results are as follows:1.The GST gene family of T.vernicifluum.can be divided into six subfamilies: ?(Phi,F),?(tau,U),?(Theta,T),?(Zeta,Z),?(Lambda,L)and DHAR(Dehydroascorbate reductase).The sequence length of most Tv GSTs gene is about 600-750 bp,the Alpha helix is the main feature of the secondary structure of Tv GSTs protein,the Tv GSTs protein of the lacquer tree is located at secretory pathway,mitochondria,chloroplast and so on,There are significant differences in cytological components,biological pathways and molecular functions between different lacquer subclasses Tv GSTs gene GO functional annotations;The different lacquer tree GST has significant differential expression in different tissues.2.The complete coding sequence of the T.vernicifluum.Glutathione S-transferase gene Tv GST7(Gen Bank accession No.: MK956145)was obtained,and its ORF is 711 bp in length,encoding 236 amino acids with the predicted molecular mass of 27.17 ku and the theoretical isoelectric point of 5.25.No transmembrane region,belongs to cytoplasmic GST;The phylogenetic tree and the three-dimensional structure analysis indicated that Tv GST7 belonges to Lambda(L)subfamily of GSTs.The results of q RT-PCR showed that the Tv GST7 gene was expressed in the roots,stems,and leaves of T.vernicifluum,with the highest expression in the stem.3.The prokaryotic expression vector was transformed into E.coli BL21(DE3).Under normal conditions,the growth of BL21/p ET28a-Tv GST7 and BL21/p ET28 a transformed cells showed no significant difference;after treatment at-20 and 50?,the growth state of the BL21/p ET28a-Tv GST7 cells was better than that of BL21/p ET28a;after treatment with 500 mmol/L Na Cl,BL21/p ET28a-Tv GST7 grew better,but BL21/p ET28 a could hardly grow;after treatment with 1 200 mmol/L sorbitol,the growth of BL21/p ET28a-Tv GST7 is slightly better than that of BL21/p ET28 a.4.The prokaryotic expression system and Ni-NTA affinity chromatography were used to express and purify the recombinant protein Lambda GST,and 27 ku of soluble protein was obtained.50 mmol/L imidazole is the best elution concentration,and the protein mass concentration was 130 ?g/m L,a single band after protein purification.Tv GST7 protein has activity on substrate DHA,specific activity is(5.36 ± 0.01)?mol/(min·mg);Tv GST7 protein has weak activity on substrate HED,specific activity is(0.01 ± 0.01)?mol/(min·mg);And Tv GST7 protein did not detect activity on CDNB,DCNB,NBC and NBD-Cl.The above research is helpful to understand the role of Lambda GST in T.vernicifluum metabolism and stress resistance,and can provide molecular biological basis for stress resistance breeding of T.vernicifluum.