Transcriptomic Analysis And Systematic Mining Of Genes Involved In The Biosynthetic Pathway Of Triterpenoid Saponins In Ilex Asprella

Triterpenoid saponins are a class of secondary metabolites, which have diverse bioactivities and are wide spread in the plant kingdom. There are various types of carbon skeletons of triterpenes, which can be classified according to the number of rings of the skeleton. Among them, tetracyllic triterpenes and pentacyclic triterpenes are the most common ones. Owing to their significant bioactivities, triterpenes are usually exploited as drugs. However, difficulties in the extraction and isolation process hinder their utilization as drug. Recently, the biosynthetic pathway of triterpenoid saponins has drawn researchers’ attention and more and more relavant papers are published. One of the most popular research topic is the biosynthesis of pentacylic triterpenes, which are represented by the oleanene-type (or the β-amyrin type) triterpenes. Meanwhile, the ursane-type triterpenes, isomer of oleanene, are poorly understood.Radix Ilicis Asprellae (Chinese name:Gangmei), is one of the most commonly used folk herb in the Lingnan area of China. By far, the most characterized chemical compounds in Ilex asprella are over30triterpenoid saponins and a few phytosterols. These triterpenoid saponins are of α-amyrin type, except for the oleanolic acid, which is of β-amyrin type. Thus,I. asprella is an ideal material for the study of the biosynthesis of triterpenoid saponins. The aim of this study is to investigate the biosynthesis pathway of the a-amyrin type triterpenoid saponins in I. asprella, using transcriptome sequencing, co-expression analysis and gene cloning. A more detailed picture of this study is presented as followed:1Transcriptome sequencing of the I. asprella rootIllumina HiSeqTM2000, which is an advancing next-generation sequencing technique, was carried out to sequenc the transciptome of the I. asprella root. A transcriptome database, which contains abundant bioimformation, was obtained, with a size of3.98Gb.51,865Unigenes were generated, among which39,269unigenes were annotated to at least one database, including Nr, Nt, Swiss-Prot, KEGG, COG and GO, accounting to75.71%of the total unigenes.2Mining of candidate unigenes relating to the triterpenoid saponins biosynthetic pathwayIn the I. asprella transciptome,272unigenes (yield of1.30%) were annotated to be involed in5terpene-relating pathways. In addition to gene annotation, homology-based BLAST and phylogenetic analysis with well characterized genes in the biosynthesis of triterpenoid saponins from other plants were also performed, and resulted in97candidate unigenes. These candidates includes22involved in the MVA pathway,23involved in the MEP pathway and26encode the synthases of several intermediates, e.g. GPP, GGPP and FPP, for the upstream pathway; and9OSCs,11CYP450s and6UGTs were found for the downstream pathway. These candidate unigenes could be submitted to further study, such as functional analysis and protein expression.3Expression patterns of candidate genes involved in the biosynthetic pathway of triterpenoid saponins10Unigenes, involved in6procedures of the biosynthetic pathway of triterpenoid saponins, were selected as target genes and submitted to gene expression study by RT-qPCR analysis, with a-tubulinas the reference gene. The result shows that:i. FPPS, SS and SM were expressed in all8tissues of I. asprella at a level similar to the reference gene; ii. Among the three OSCs determined in this study, CL3079.contigl expressed in all tissues at a relative high level compared to the reference gene, whereas the other two expressed at a very low level; iii. The2CYP450s and2UGTs expressed at a low level. Besides, the concentrations of total saponins in the8tissues of I. asprella were determined using chromometry. Relationship between the gene expression levels and the chemical metabolism was further discussed:(i) Triterpenoid saponins are probably synthesized at various tissues of I. asprella;(ii) Triterpenoid saponins are mainly stored at the roots and leaves;(iii) Stem might be responsible for triterpenoid saponins transportation, for there is little storage in it.4Gene cloning and functional analysis of several amyrin synthase9candidate OSCs were found in the transcriptome of the I. asprella root. Among them, unigenel015was annotated as a-amyrin synthase, while CL3079.Contigl and CL481.Contigl (designated as IAAS3079and IaAS481) were annotated as β-amyrin synthase, which contain a full length of CDS. Utilizing T-A cloning and the GATEWAY technology, expression vectors of IaAS3079and IaAS481with sepecial tag were constructed and transformed into yeast. The Western-blot results showed that IaAS3079was successfully expressed in yeast, while IaAS481wasn’t. Furthermore, cell extract of the yeast harboring, in the two expression vectors, respectively, were analyzed by GC-MS. Result showed that both a-amyrin and β-amyrin were found, but with different product ratio, indicating that both Ia3079and IaAS481were multi-functional amyrin synthases. The product catalyzed by IaAS3079is mainly a-amyrin with the yield of80%; while that for IaAS481is mainly β-amyrin with the yield of95%. In addition, by utilizing RACE technology, the missing fragment at the3’end of unigene1015, about800bp, was successfully amplified, while that in the5’end wasn’t.In conclusion, a series of candidate unigenes involved in the biosynthetic pathway of triterpenoid saponins have been identified by combination of various modern biological technologies. Ursane-type triterpenoid saponins, which are the main chemical composition of I. asprella, have anti-cancer and anti-virus activities. Thus, they are considered to be the pharmacodynamics substance of I. asprella. The study of the biosynthesis of triterpenoid saponins at the molecular level provides a basis for future biological metabolism engineering of I. asprella, and opens a new door for the germplasm biochemistry study of I. asprella.