Study On The Synthesis Mechanism Of Isoforskolin,an Active Component Of Coleus Forskohlii Based On Transcriptase Sequencing

Coleus forskohlii(Willd.)Briq.,also known as Qiao Ruisu,is one of Labiatae(Coleus Lour.)Plants,mainly produced in India,Nepal,Pakistan,the United States,Sri Lanka,Myanmar,Thailand and other South Asian countries;a rare medical plant defined by botanists distributing in Yunnan,Fujian,Taiwan and other places in China.The herb used for the treatment of asthma,cough and other diseases in Folk,because of the Significant effect,the plant known as "God medicine".Modern pharmacological studies demonstrate that the active substance being Labdane-type diterpnoids.The active ingredient of our country C.f is isoforskolin,as an indicator component.In view of the unique effect of efficacy of C.f,our laboratory cooperate with the Hubei Furen Pharmaceutical Company to implement the combination of production and research,to develop a new drug named “Coleus capsules”based on this trasitional medicine as monarch drug,compatibility of Angelica decursiva and liquorice,and this drug get a production license(NO.2011S00365).In order to Preserve the rare species of wild C.f resources and solve the problem of supply and demand,Hubei Furen Pharmaceutics Co.transplanted Coleus forskohlii from Huize of Yunnan Province to Tongcheng of Hubei Province and highly spread its medical application in markets.However,the low content of isoforskolin is the key problem in medicine quality control of Coleus forskohlii production.In this paper,we focused of the mechanism of the diterpenoids biosynthesis of C.f.In order to increase quality of this medical germplasm and finally to improve the quality of this herbs which grow in Tongcheng.Besides,our lab also found that the rate of synthesis diterpenoid increased after coleus were exposed in UV light and tried to analyzed the mechanism behind this phenomenon.The main findings are as follows:1.summarizing and analyzing Coleus forskohlii transcriptome data based of Illumina Hi SeqBasing of C.f leafs and roots as test materials,which acquisition in late October,the 36 Gb transcriptome data were obtained and performed by Hi Seq technique,include 422761 high quality sequences.The assembled Unigene gene was predicted by ESTscan(V2.1)analysis to obtain CDS sequence and Protein sequence.Using Trinity and TGICL method from the beginning of assembly,sequence splicing and de-redundant processing.A total of 291,061 unigenes were assembled with the average unigene size of 629 bp;and140,228(48.19%)unigenes were assigned,The 140,228 assigned Unigene was analyzed by GO,COG cluster analysis and KEGG metabolic pathway86,578 Unigene genes were obtained from COG function annotation and classified as 25 functional categories.A total of 186,957 Unigene genes were obtained by GO Functional annotations were classified into 49 GO secondary functional categories;a total of 6,616 Unigene could be assigned to 261 metabolic pathways.Therefore,this study lays the foundation for the cloning,sequence analysis and expression of terpene synthase gene,which is the active component of C.f.,and the 23 terpene differences in roots and leaves provide the basis for the gene control targets of traditional breeding and transgenic breeding.2.Cloning,Sequence Analysis,functional verification and Expression ofthe C.f terpene synthase genes,which based on High Throughput SequencingBased on the above high-throughput gene sequencing study,the full length c DNA sequences of three terpenoid synthases were obtained by using RACE technique,and analyzed by BLAST and protein sequence analysis.The results show:(1)The full-length Cf GCS gene is 2059 bp,which contains 235 bp 5'untranslated region,1632 bp open reading frame and 192 bp 3' noncoding region,encoding 544 amino acids.The construction of expression vector by using E.coli expression system enables access to Cf GCS protein.The physical and chemical properties of Cf GCS are predicted.The relative molecular mass of Cf GCS was 63690.84;isoelectric point is 5.23;Molecular formula C2868H4438N738O848S27.q PCR indicated that Cf GCS had a high expression level in the leaves.(2)The full-length Cf VPS gene is 1874 bp,including the 5 'untranslated region of 116 bp,the open reading frame of 1647 bp,the 3' untranslated region of 111 bp,the relative molecular mass of Cf VPS is 63608.40,the isoelectric point is 5.06,and the molecular formula C2859H4412N744O852S24.The expression vector PET28-Cf VPS was constructed with Cf VPS as target gene,and expressed in Escherichia coli at 32 ? for 6 hours.Cf VPS was involved in the synthesis of plant monoterpene compounds,and the distribution of glandular hairs was the main site of monoterpene synthesis.The results of q PCR also showed that Cf VPS had the highest expression in leaves.(3)The Cf GAS gene molecular weight is36895.20,isoelectric point6.37 and molecular formula C1672H2575N437O482S12.The full-length Cf GAS gene is 1234 bp,which contains 72 bp 5 'untranslated region,Compared with Camellia sinensis,Salvia miltiorrhiza,sweet potato,cannabis,tobacco,white winter solanata and wheat,it was found that Cf GAS protein was close to that of Salvia miltiorrhiza.At the same time,we constructed the recombinantvector of GAS by Gateway,and provided the experimental basis for the subsequent rooting of Agrobacterium tumefaciens.The results of q PCR showed that Cf GAS was involved in the synthesis of gibberellin.Gibberellin was a diterpenoid plant hormone,which played an important role in the whole growth of plants and distributed in all tissues.3.Study of the molecular Mechanism behind how UV Irradiation can increase the Content of effective components in coleusBasing on the inspiration of critical genes of diterpenoid synthetase,this paper has investigated the biosynthesis mechanism of what effect of UV had on diterpenoid synthesis.From the result we can know that UV light can increase the rate of diterpenoid synthesis effectively,53% exactly.Through method of q PCR,we acquired the efficiency of gene expression of terpenoid materials and the results shows that all of TPS1,TPS2,TPS3,TPS4 and TPS14 synthase can increase the rate of synthesis isoforskolin while TPS15,gibberellin synthase and kauri will decrease the rate.Therefore,this paper provides an excellent scientific prove of how to increase the effective content of medicine and explored new ways to improve the quality of Chinese herbal medicines which come from standardized cultivation.