Studies On The Medicinal Active Ingredients Of Hawthorn And S. Sylvestris And Their Key Synthetic Enzyme Genes

Cornus officinalis Sieb.et Zucc.(Cornus,Cornaceae),a deciduous tree or shrub,is used as traditional Chinese medicine for its dry pericarp which has the efficacy of tonifying the liver and kidney,regulating immune,nourishing vitality and conserving deficiency.The main active ingredient of C.officinalis is iridoid glycosides(CIGs),e.g.loganin and morroniside.It has broad pharmacological activities such as neuroprotection,treating diabetic,nephropathy,preventing inflammation and apoptosis.Cornus controversa has close phylogenetic relationship with C.officinalis,and is usually used as ornamental plants.However,little is reported on its medicinal values and active ingredients.At present,the studies of C.officinalis mainly concentrated in germplasm resources,breeding and cultivation of excellent varieties,and physiological and genetic diversity,etc.There are no studies concerning about the genomic data and related molecular studies.Also,it still remains unknown how loganin is synthesized and accumulated in Cornus plants.In recent years,due to the gradual marketization of transcriptome sequencing technology,the genetic information data of specific species can be easily obtained by transcriptome data,which can lay foundations for the research about the synthesis pathway,gene clone and expression of active ingredients.In this study,we chose C.officinalis and C.controversa as materials.The germplasm characteristics of fruits of the two species during growing stage were observed.The total sugar,total organic acid,total cornel iridoid glycosides and loganin content were compared between the species.The Illumina high-throughput sequencing technology is adopted to establish C.officinalis transcriptome database.The key genes involved in the synthesis pathway of loganin were cloned.Furthermore,the expression of these genes was detected by Real-time PCR.Together with the dynamic changes of loganin content,the synthesis mechanism and accumulation pattern of loganin were preliminary revealed.The primary results of our study are as below:1.The germplasm characteristics of the fruits of C.officinalis and C.controversa in growing stage were comparatively analyzed.The results showed that the size,the hundred-seed weight and moisture content of C.officinalis were significantly higher than those of C.controversa(P<0.05),while the pericarp rate of former was slightly lower than the later.In the early stage of fruit development in C.officinalis,the kernel grows fast and turns hard,and in the later period the pulp increases significantly,which is speculated to synthesize metabolites.In the whole growth period of the two species,the fruits size,hundred-seed weight,and pericarp rate gradually increased along with the growth stage,and the moisture content was relatively stable.2.The ultraviolet spectrophotometer and HPLC method were used to measure the total sugar,total organic acid,total cornel iridoid glycosides and loganin content of C.officinalis and C.controversa in different stages and different parts.The results showed that the contents of all the component in fruits are higher than those in leaves and flowers of C.officinalis,and each component increased significantly during the fructescence(P<0.05).The contents of total sugar and total iridoid glycosides in fruits of C.controversa are higher than those in leaves and flowers,meanwhile the contents of total organic acid and loganin in fruits are less than those in leaves and flowers.The content of all these components of fruits and leaves decrease while the fruits grow to full maturity.C.controversa has a very low loganin conten,which is also obviously lower than that of fruits of C.officinalis(P<0.01).3.The pericarp of C.officinalis was chosen as material,and the high-throughput sequencing technology was used to establish the C.officinalis transcriptome database.44,718,948 clean reads and 71,842 unigenes were obtained.Aligning with other databases,31,780 unigenes were annotated.Through the alignment with KEGG database,9,978 unigenes were mapped into 268 metabolic processes,of which 105 unigenes were annotated to terpenoid biosynthesis.There were 16 key enzyme genes in the synthesis pathway of loganin(including G10H,SLS,etc).Additionally,13,719 SSR loci were discovered by filtering with MISA software.With the alignment of BlastX searching and Nr database in NCBI,1,233 transcription factors were acquired.4.The conserved sequences of G10H and SLS in the C.officinalis and C.controversa were cloned respectively;the conserved sequences of 18S gene in C.controversa were cloned;Furthermore,the expression of G1 OH and SLS in C.officinalis and C.controversa were detected using Real-time PCR.Together with the loganin content of the two species,we primarily found that the expression of G10H is positively correlated to loganin content,while the expression of SLS is negatively correlated to loganin content.But the correlation is not significant,because the metabolic pathway network of loganin might be complex,these two genes might be involved in the synthesis of other components.In this study,we compared the interspecific differences of characters and the content of active ingredient between C.officinalis and C.controversa.Using the transcriptome data of C.officinalis,we cloned the key genes involved in the synthesis pathways of loganin in the two species.Furthermore,we primarily analyzed the correlation between the expression of G10H and SLS and the content of loganin.These results will lay foundations for future research investigating the synthesis and accumulation of cornel iridoid glycosides.