Cloning And Expression Analysis Of Key Enzyme Genes Of Triterpenoid Biosynthesis Pathway In Tripterygium Wilfordii Hook.f.

Tripterygium wilfordii is an important medicinal plant that has anti-cancer,anti-inflammatory and reg?lating the immune system effect,also has strong insecticidal activity.Celastrol is the most representative triterpenoid in T.wilfordii which has high pharmacological activity for medical application and potentional development value.Squalene synthase?SQS?,squalene epoxidase?SE?and oxidosqualene cyclase?OSC?considered to be the key rate-limiting enzymes in the triterpenoid biosynthesis pathway.In this paper,we cloned the one squalene synthase gene,two squalene epoxidase genes and four oxidosqualene cyclase genes from T.wilfordii.And then we analyzed the predict amino acid sequences by bioinformatic analysis.Further,gene expression patterns in different tissues and the expression level of genes in T.wilfordii hairy root after treatment by methyl jasmonate?MeJA?were detected by real-time PCR.At the same time,HPLC detected changes of celastrol content.The main results are as follows:1.The open reading frame?ORF?of the TwSQS gene is 1242 bp and encode 413 amino acids.The truncated TwSQS gene of the C-terminus last 28 amino acids was ligated into the pET-30a?+?vector and transfected into the E.coli Transetta?DE3?.After induced by IPTG,SDS-PAGE analysis showed that TwSQS was obtained soluble protein,with a molecular weight of around 49.41 kDa.In vitro enzyme reactions demonstrated that TwSQS protein could catalyze the reaction of FPP form to squalene under the coordination of Mg²⁺and NADPH.TwSQS is expressed constitutively in different tissues and has the highest expression level in roots.After induced by MeJA,the expression level of TwSQS rapidly increased in early stage,reached the highest value after induction 6 h,and gradually decreased with the prolongation of induction time,but the expression level was still significantly higher than that of 0 h.2.The ORF of TwSE1 and TwSE2 were 1578 and 1584 bp,which encoding 525 and 527amino acids.Two amino acid sequences from TwSE1 and TwSE2 shared 76.18%similarity,but were highly divergent in N-terminal regions.TwSE1 and TwSE2 genes have also constitutively expressed in different tissues of T.wilfordii.TwSE1 expressed highest in flowers and lowest in roots,but TwSE2 expressed highest in flowers and lowest in old leaves.The expression of TwSE2 was lower than that of TwSE1 in all tissues.MeJA treatment enhanced the TwSE1 and TwSE2 expression level in hairy root.The expression level of TwSE1and TwSE2 has an increasing tendency in the early stage,then decreased in the middle stage and increased in the late time.The highest expression was reached after the treament of 3 h,then decrease,and the expression level increased rapidlly after 12 h.In same treatment condition,the increase of TwSE2 gene expression level was less than that of TwSE1 gene.3.The ORF of Tw OSC1,Tw OSC2 and TwOSC3 were all 2289 bp and encoded 762amino acids.The TwOSC4 ORF was 2280 bp and encoded 759 amino acids.Multiple sequence alignments of four TwOSC genes discovery that they had high similarity of 72.06%.The TwOSC1 gene has a very strong tissue-specificity and only expressed in the root.However,TwOSC2,TwOSC3,and TwOSC4 were expressed in all different tissues.TwOSC2had the highest expression in roots,and the other four tissues expression levels were significantly lower than those in roots.TwOSC3 had the highest expression in old leaves,and the lowest expression in roots and stems.There was no significant difference in the expression of TwOSC4 among roots,stems and flowers that higher then leaves.Four TwOSC genes are all differently responded to MeJA stimuli.After the treatment of MeJA the expression of TwOSC1 gene did not change significantly in the first 6 h.As the stimuli time increases,the expression level of TwOSC1 gradually increases.The TwOSC2 gene reached the highest expression level after treatment of 6 h,as the istimuli time prolonged the expression level gradually decreased.MeJA inhibited the expression of TwOSC3 gene,and the expression level has a transiently increased within 3 h,then decreased gradually.After treatment of 12 h,the expression level increased,but it was still lower than that before uninduced.After treatment by MeJA the expression level of TwOSC4 gene showed a trend of first increase,then decrease and then rise.After treatment of 12 h,the expression level increased significantly.4.HPLC detection of celastrol content in different tissues only detected in the T.wilfordii root.After treatment of MeJA,the content of celastrol in T.wilfordii hairy roots increased.The content of celastrol was no significant increase in the first 6 h,then increased significantly and reached the highest value at 24 h.After treatment of MeJA,the change of TwOSC1 expression was consistent with the changes of celastrol content.The TwOSC1 was also only expressed in T.wilfordii root,suggesting that TwOSC1 may be related to the synthesis of celastrol.