Cloning And Analysis Of CfCPS Gene And Mechanism Underlying Isoforskolin Improvement By Paclobutrazol Based On CDNA Library In Coleus Forskohlii

Coleus forskohlii(Lamiaceae,Syn.Plectranthus barbatus Andr.)grows perennially over the tropical and subtropical regionsof Asia(India,Pakistan,Sri Lanka,China,south of Arabian Peninsula,etc).It grows sparsely in China’s Yunnan,Guangdong,Fujian and Taiwan and is defined as a rare plant by botanists.Therefore it is labelled as Coleus forskohlii herb,recorded in Volume 66 of the National Herb Journal "Flora of China" and "Yunnan Drug Standards".It is commonly used for treating asthma,cough and other diseases.Since the effect is obvious,it is known as the "panacea".Modern scientific research shows the herb has great anti-tumor effect with its active substance being Labdane-type diterpenoids.In light of its unique medical effects,Hubei Furen Pharmaceutical Corporation developed a new drug named “Coleus capsules” based on this traditional Chinese medicine,and this drug have obtained production licenses(NO.2011S00365).In order to preserve the rare species of wild coleus forskolin resources and to solve the problem of supply and demand,Hubei University of Chinese Medicine and Hubei Furen Pharmaceutical Company cooperate on scientific and industrial levels.We have this herb transplanted from Yunnan Huize coleus forskolin to Tongcheng County and begin anormalized plantation.This paper applies biotechnology to artificially synthesize high quality herb species since this method require gene regulation of medicinal plants and careful cultivation of plant herbs.We found that the main biologically active compounds of Coleus forskohlii are diterpenoids.This study has constructed a full-length normalized cDNA library to find a diterpenoids biosynthetic enzyme unigene by EST analysis.This study provided molecular biology research to reveal the mechanisms of the synthesis and accumulation of active components of coleus forskolin,thus paving the way for future drug development and quality control of artificial production of coleus forskolin.All these researches provide a scientific basis to analyze metabolism and accumulation mechanisms of main active ingredient in planted coleus forskolin so as to increase quality of this medical germplasm and finally to improve the quality of planted herbs.The main findings are as follows:1.Construction of coleus forskolin full-length normalized cDNA libraryThe mixture of various tissues of C.forskohlii plants was used as starting material for cDNA library construction.The double-strand cDNA was amplified by long-distance PCR,and appeared as a 0.5-3 kb smear on the agarose gel(1.2%).The titer of the original library was approximately 1.6×106cfu/ml with recombination percentage of 93.05%.The quality of the resulting library was assessed by electrophoresis of PCR products of randomly picked30 clones.The insert size of cDNAs ranged from 1 to 3 kb,suggesting relatively long cDNAs contained in the library.2.EST sequencing and bioinformatics analysisA total of 4224 cDNA clones were sequenced and 2394 unigenes were assembled with the average unigene size of 753 bp.A total of 2100(87.7%)unigenes were functionally classified using gene ontologies,and 1716(71.7%)unigenes were assigned to establish pathway associations in KEGG.Notably,64 unigenes putatively participated in the biosynthesis of secondary metabolites,in which 17 unigenes were identified that might be involved in the biosynthesis of the terpenoid backbone and monoterpenes,diterpenes,and triterpenes.Eleven unigenes,corresponding to 11 EC numbers were identified possibly involved in terpenoid backbone biosynthesis according to KEGG mapping.Also identified in the cDNA library were three enzymes involved in diterpene biosynthesis(ent-copalyl diphosphate synthase,ent-kaurenoic acid hydroxylase,and CYP82G1),two monoterpene synthases(myrcene/ocimene synthase and neomenthol dehydrogenase),and one enzyme involved in triterpenoid biosynthesis(β-amyrin synthase).3.Cloning Coleus forskolin CfCPS genes,functional verification,sequence analysis and expressionOn the basis of EST sequencing,choosing from the copal unigene ene pyrophosphate synthase(CPS)fragment gene sequence by using RACE technology gains a full-length cDNA sequence.The result shows that the full-length gene CfCPS is 2418 bp.The construction of expression vector by using E.coli expression system enables access to Cf CPS protein.CfCPS genes.Bioinformatic analysis of CfCPS revealed that the full length sequence is2418 bp,encoding 805 amino acids,molecular weight of 92,235,the formula C4166H6415N1101O1219S29,,PI 5.70.Multiple alignments withRosmarinus officinalis、Salvia miltiorr、Marrubium vulgare、Castananea mollissima、Prunus persica)、Scoparia dulcis、Solanum lycopersucum、Zea mays showed that they have a high homology.Further studies showed that the transmembrane region and CfCPS has no signal peptide with a soluble protein feature.By qPCR method,we detected Coleus forskolin CPS gene expression has a higher gene expression in roots.4.Molecular mechanism underlying the increment of isoforskolin by paclobutrazolMolecular mechanisms of the increment of isoforskolin by paclobutrazol treatment were illustrated based on the key genes obtained above.Plant shape of Coleus forskolin was greatly improved by paclobutrazol with the concentrain of 100mg/L,as well as isoforskolin content(up to 53%)in roots,rhizomes,and stems.The gene expression levels of leaves and roots of plant treated by paclobutrazol were determined by qPCR,and the results showed that the expressions of ACCT,HMGR,DXR,IDI,GGPS,and KAO gene were markedly induced.This results sugest that the improvement of isoforskolin biosynthesis by paclobutrazol may mainly resulted from the up regulation of ACCT,HMGR,DXR,IDI,GGPS,and KAO gene.