Cloning And Functional Study Of The Diterpene Synthase Genes From Isodon Rubescens

Isodon rubescens is a perennial herb of the genus Isodon.The dry aerial part of the plant used for traditional drug.It is commonly used to treat sore throat,chronic hepatitis,rheumatoid arthritis,bronchitis and other diseases.In recent years,its anti-cancer activity has attracted much attention.The main anticancer active substances are components such as Rubescensine A and Rubescensine B,and belong to the enantiomeric kauritan type diterpenoid compound.However,the traditional production model base on plant has greatly limited its promotion.The method of producing medicinal active ingredients by synthetic biology is expected to break this limitation,but this method must be base on reveal the metabolic pathway in plants.In this study,molecular metabolic methods were used to analyze the metabolic pathways of active diterpenoid active constituents in Rubescens.In this study,the diterpene synthase genes are cloned from I.rubescens and the proteases are functionally identified.The main research contents and conclusions are as follows:1.Through the transcriptome sequencing and data analysis of the leaf tissue of Rubescens,11 candidate diterpene synthase genes were screened,and the full-length cDNA of these genes was cloned from cDNA library.The DNA sequence analysis of the gene obtained the sequence of amino acids,molecular weight,isoelectric point and transit peptide of the corresponding protein.IrCPS1,IrCPS2,IrCPS3,IrCPS4,and IrCPS5 contain a "DXDD" sequence and belong to the type II terpene synthase.IrKSL1,IrKSL2,IrKSL3,IrKSL4,IrKSL5 and IrKSL6 contain a "DDXXD" sequence and belong to the type I terpene synthase.Phylogenetic analysis further clarifies the possible biochemical functions of individual proteins.The protein function was predicted as follows: IrCPS1 and IrCPS2 catalyze GGPP to produce normal-CPP,IrCPS4 and IrCPS5 catalyze GGPP to produce ent-CPP,and IrCPS3 has no biochemical function due to amino acid conserved site mutation.IrKSL1,IrKSL3,IrKSL6 can catalyze normal-CPP,IrKSL2,IrKSL4,IrKSL5 can catalyze ent-CPP.Tissue-specific expression analysis revealed that both IrCPS1 and IrKSL1 genes are mainly expressed in roots and may be responsible for the metabolism of rosin-type diterpenoids in roots.In contrast,IrCPS4,IrKSL2 and IrKSL4 are mainly expressed in leaves,possibly The important role of the amyloid-type active substance in the metabolism of oridonin is important;the relative expression of IrCPS3 in each tissue is very low,and it is speculated that there is no biochemical function.2.Five type II diterpene synthases were expressed by prokaryotic expression system,and the expressed protein was purified by affinity adsorption.The protein function was identified by in vitro enzymatic reaction and GC-MS.The results showed that IrCPS1 and IrCPS2 could catalyze GGPP to generate normal-CPP,IrCPS4 and IrCPS5 could catalyze GGPP to generate ent-CPP,and IrCPS3 had no biochemical activity.This result is consistent with the functional prediction results of the five type II diterpene synthases using bioinformatics methods and phylogenetic tree cluster analysis.It is predicted that IrCPS3 may not be biologically active because its key conserved sequence "DXDD" is mutated to "DXND"."DXDD" is considered to be a key site for the initiation of GGPP into a loop.Therefore,we used a sitedirected mutagenesis technique to reverse-mutate the conserved site of IrCPS3 and transform it into "DXDD".However,in vitro enzymatic reaction revealed that the mutant IrCPS3-N333 D still could not catalyze the GGPP production product.Therefore,we speculate that there are other key sites in addition to "DXDD" that cause IrCPS3 to lose its function.To find out these sites to further understand the catalytic mechanism of CPS genes,further analysis and screening and mutation studies are needed.3.The six type I diterpene synthase genes were expressed by prokaryotic expression system,and the expressed protein was purified by affinity adsorption,and the protein function was identified by in vitro enzymatic reaction and GC-MS.The results showed that IrKSL1 and IrKSL3 could catalyze the formation of secondary tanshinone ketone by normal-CPP,and isopimaradiene by normal-CPP catalyzed by IrKSL6.IrKSL5,like AtKS,catalyzes the creation of ent-kaurene by ent-CPP.In addition to the trace amount of ent-kaurene,IrCPS4 with IrKSL4 is mainly products ent-atiserene.IrKSL2 catalyzes ent-CPP products isopimaradiene by consistent with the retention time.Mass spectrometry ion fragments are highly similar,but isopimaradiene is opposite to the 11-and 20-position C atom conformations of IrKSL2,so it is speculated that these two substances are isomers.Further structural identification is required to determine the absolute conformation of the IrKSL2 product.IrKSL6 can catalyze the formation of different products of normal-CPP and ent-CPP,respectively,but the enzymatic kinetics test shows that the affinity of IrKSL6 to substrate normal-CPP is significantly higher than that of ent-CPP.Kcat/Km showed that the efficiency of normal-CPP catalyzed by IrKSL6 is about 1127 times that of ent-CPP.4.Two genes,IrKSL3 and IrKSL3 a,were cloned from the leaves of Rubescens leaves using the same pair of primers.Sequence alignment revealed that the two genes differed only by a length of 18 bases,encoding six amino acids "ILTHYR",and the other sequences were identical.Further analysis of its genomic sequence revealed that the two cDNAs were derived from the same gene in the genome and were generated by alternative splicing through different 3’ splice sites.Functional studies have found that IrKSL3 a can not only produce a small amount of the product of IrKSL3,but also a more important compound,isopimaradiene.Next,the reasons for this difference were analyzed by site-directed mutagenesis and protein three-dimensional structural homology modeling.It was found that the functional difference between the two proteins may be caused by a change in the number of amino acids resulting in a change in the position of the tryptophan at the lower end of the E-helix.By cloning the diterpene synthase genes from I.rubescens and identifying their functions,the first two steps of the downstream biochemical pathway of diterpenoids in I.rubescens were identified.This study lays an important foundation for the functional research of the downstream CYP450 genes and finally reveal the entire diterpene metabolic network of I.rubescens.