Cloning And Positive Selection Analysia Of Squalene Synthase Of Hemsleya Chinensis In Cucurbitaceae

Squalene synthase(SS)is a key enzyme in the plant triterpenoid biosynthesis pathway,which is responsible for condensing two molecules of farnesyl pyrophosphate(FPP)into a squalene(SQ).SS has a crucial role in the follow-up products in the triterpenoid synthesis,and the activity of SS usually determines the subsequent products in triterpenoid saponins pathway.In recent years,people pay more attention to how to improve the activity of SS and then increase the content of the triterpenoid saponins in plants.In this study,with the 3'RACE and 5'RACE(rapid amplification of cDNA ends)method,the full-length cDNA sequence of the squalene synthase was isolated from Hemsleya chinensis for the first time,and the bioinformatics analysis and positive selection analysis of Hemsleya chinensis were carried out,which laid an important foundation for SS in the triterpene synthesis in Cucurbitaceae.The main results of this research are as follows:1.HcSS gene cloning: with the method of 3'RACE and 5'RACE,Hemsleya chinensis SS gene cDNA sequence was cloned,and we obtained1200 bp and 630 bp respectively and finally,we got the full length sequence of SS 1609 bp,includind anopen reading code sequence 1254 bp,and encoding417 amino acid residues,and its molecular weight size is about 47.81 kD.2.HcSS gene sequence analysis: with NCBI Blast comparison,it was found that the nucleotide sequence of SS gene was 92% homologous to the SS nucleotide sequence of Gynostemma pentaphyllum,and the amino acid sequence homology was 93 %.3.The positive selection analysis of HcSS: with the Bayesian method to reconstruct the phylogenetic tree of SS in terrestrial plants,we found that relationships between Hemsleya chinensis and Cucurbitaceae plants such as Trichosanthes rubriflos,Gynostemma pentaphyllum,Siraitia grosvenorii,and the Momordica cochinchinensis were closed,and the posterior probability among them are all 1.00.The cDNA sequence of Hemsleya chinensis SS is of the highest homology with the squalene synthase gene of Cucurbitaceae,which was consistent with the predicted results.The adaptive evolution of Hemsleya chinensis SS and 59 plants SS was studied and analyzed by PAML4 software,using the site model,branch model and branch-site model.The results showed that the evolutionary relatively conserved SS gene was under positive selection,and we found that there are 45 positively selected sites in the site model and 12 in the branch-site model,which were mostly concentrated in the secondtransmembrane region and its two sides.Positively selected sites 189 S,196S,265 T,389P,390 T,407S,410 R and 414 N sites are likely to involved in biochemistry process and may be related to the activity of HcSS.Especially the positively selected site 196 S is of the largest correlation of SS enzyme.These positively selected sites will play important roles in exploring the key functional sites and structures of plant SS.In the future,site-directed mutation experiments will be carried out to verify these important sites.In this study,we successfully cloned the full-length cDNA sequence of the Hemsleya chinensis SS gene and obtained the positive selection analysis of SS in Hemsleya chinensis and 59 terrestrial plants,which lay an important foundation for exploring the correlation between the positive selection and function of the key enzyme SS in the triterpene synthesis pathway of Cucurbitaceae.In this study,the adaptive evolution of SS was studied by molecular evolution method.In order to understand the molecular mechanism of triterpenoid saponin biosynthesis pathway and its regulation,the triterpenoids biosynthesis will be controlled by artificial regulation at the molecular level,improving its active ingredient.It is of great significance in triterpenoid saponin biosynthesis.