Cloning And Functional Analysis Of β-amyrin Synthase Genes Associated With Oleanolic Acid Biosynthesis In Gentiana Straminea And Its Somatic Hybrid

Recently,the research hot spot of plant secondary metabolic engineering is turned into cloning and transfomation of genes coding key enzymes,of which the overexpression has been one of major strategies to increase the yield of target metabolin in this area.However,the related genes in the pathway were still known a little,according to the complexity of the relationship between metabolic pathways,the limited modus operandi to character functional gene.At present, the biosynthetic pathway of terpene was investigated at gene level and several genes were cloned. However,it is just started the research of gene discovery in Chinese crude drug.Gentiana straminea Maxim.(Mahuaqinjiao) is an important medicinal plant in China,and has anti-hepatitis activity,while its natural resources were threatened by excessive exploitation. Bupleurum scoronerifolium is one of the famous Chinese medicinal materials with an anti-cold activity.Somatic hybridization has been proven to increase the contents of effective components in the somatic hybrids and realize the transfer of the genes related with the medicinal components in interspecific,intergeneric or even inter-familial plants.The somatic hybrids of B.scoronerifolium and G.straminea with higher content of oleanolic acid had already been obtained in our lab.Theβ-amyrin synthase gene,which plays an important role in the synthesis of oleanolic acid,has a different expression in the parents and the hybrid(B.scoronerifolium with one copy,G.straminea with two and hybrid with two).However,cloning,function and existing way ofβ-amyrin synthase gene in the parents and the hybrids had short of systemic and comparative analysis.Oleanolic acid,having anti-hepatitis activity,belongs to oleanane type of pentacyclic triterpenoid.β-amyrin should be oxygenized and carboxylated at the C-28 position to form oleanolic acid.β-amyrin synthase,a member of oxidative squqlene family,is the key enzyme to synthesizeβ-amyrin and its downstream product oleanolic acid.In this work,the clone and functional analysis ofβ-amyrin synthase gene in the biparents and hybrids between B.scoronerifolium and G.straminea was studied.We isolated and clonedβ-amyrin synthase genes from the biparents and hybrid clones for the first time;verified the functiona of GsAS1and GsAS2 by E.coli,yeast;realized the transformation of GsAS1and GsAS2 in embryogenic calli of G.straminea;obtained many transgenic plants with higher content of oleanolic acid.It was laid a theoretical foundation for the advanced research about the regulation mechanism of oleanolic acid metabolism.On the other hand,we analyzed systematically the form existed and expression pattern ofβ-amyrin synthase gene in the somatic hybrids;investigated the relation between the gene expression level and the content of oleanolic acid;compared the difference of expression of the genes related with the oleanolic acid metabolic pathway under methyl jasmonic acid treatment; found the overexpression ofβ-amyrin synthase gene with the accumulation of oleanolic acid under methyl jasmonic acid treatment.In addition,we separated and identified the volatiles organic compounds in Chinese herb Saussurea involucrate and Saussurea lacostei.Two unknown components were detected by modified P&T-GC-MS which was laid a foundation for the exploitation of Chinese crude drug resources.The main process and results of this research were as follows:1.Cloning ofβ-amyrin synthase gene related with oleanollc acid metabolismNine full-length DNA ofβ-amyrin synthase genes were isolated by RACE-PCR from G. straminea,B.scoronerifolium and their somatic hybrids(2Q,3Q,3Q1'),named GsAS1(GI: FJ790411),GsAS2,Bs AS,GsAS1a,GsAS1b,GsAS1c,GsAS2a,GsAS2b and GsAS2c respectively. Their open reading frame consists of 2268bp,2277bp,2289bp,2268bp,2277bp,2268bp,2277bp,2268bp,2277bp respectively,and predicted to encode a 755,758,762,755,758,755, 758,755,758 residue protein with molecular mass 88.0 kD.Their protein sequences shared the characteristic sequence of oxidative squqlene family,which contains 4 copy of QW reading frame[(K/R)(G/A)XX(F/Y/W)(L/I/V)XXXQXXXGXW]and 1 copy of DCTAE reading frame. Their nucleotide and amino acids sequence homology were as following:GsAS1/GsAS2 were 81.73%and 81.13%,BsAS/GsAS1 were 78.9%and 79.79%,BsAS/GsAS2 were 83.49%and 89.11%,GsAS1a/GsAS1b/GsAS1c were 100%,GsAS2a/GsAS2b/GsAS2c were 99.6%and 100.0%.Compared with the nucleotide and amino acids sequence of the 9 genes above, nucleotide and amino acids homology were 91.3%and 90.4%respectively.The phylogenetic analysis showed that all genes above are closely related to other plant OSCs,and particularly to the mono-functionalβ-amyrin synthases,while their amino acids sequence had higher homologous with Panax ginseng,Bupleurum Chinense,Artemisia annua and Aster sedifolius.GsAS2,GsAS2a,GsAS2b,GsAS2c didn't contain any intron by PCR,with their genomic DNA as the template using primers mentioned above.The analysis of nucleotide sequence indicated that GsAS1a/GsAS1b/GsAS1c(from hybrids) were consistent with GsAS1,GsAS2b were consistent with GsAS2(from G.straminea) respectively,while GsAS2a and GsAS2c had small mutation in GsAS2 with 1 and 2 bases respectively.Therefore,the nucleotide sequence homology analysis among above genes mentioned indicated that they all have the function ofβ-amyrin synthase.2.Expression profiling of the genes related with oleanolic acid synthesis pathway1) Expression pattern of GsAS1 and GsAS2The results of RT-PCR and Real-time PCR analyse showed that the expression of both GsAS1 and GsAS2 in the leaf was higher than in the roots and shoots.GsAS1 and GsAS2 are expressed in a tissue-specific manner,with its expression in the leaves being～2.8-fold and～5.1-fold than that in the roots,and～4.5-fold and～4.2-fold than that in the stems.2) Expression of the genes related and the content of oleanolic acid in the hybrids and the biparentsThe expression levels ofβ-amyrin synthase(AS),farnesyl pyrophosphate synthase(FPPS), squalene synthase(SQS),squalene epoxidase(SE) and cycloartenol synthase(CAS) in hybrids 3Q,3Q1',2Q and both parent lines were analyzed by RT-PCR.The results showed that the expression level of AS and SQS is 2Q< biparent lines<3Q=3Q1' and the expression level of CAS is 3Q1'90kDa) than expected due to some post-translational modification.2) Functional verification in Pichia pastorisThe yeast expression vectors pPICZA-GsAS1 and pPICZA-GsAS2 were constructed and transformed into Pichia pastoris X-33.SDS-PAGE analyze detected an 88kDa band in transformed P.pastoris strains which identity toβ-amyrin synthase,proved that GsAS1 and GsAS2 are functionalβ-amyrin synthase gene.GC-MS analysis showed the positive transformed P.pastoris strains had an identical product at 15.20min withβ-amyrin synthase and shared the same construction withβ-amyrin synthase(m/z 426[M+];411[M+-CH3];393 [M+-H2O-CH3];218(C-ring fragment peak);203[m/z 218-CH3]),while the control strain did not have this product.The results proved GsAS1and GsAS2 can be expressed in P.pastoris and its product has the identity bioactivity withβ-amyrin which is synthesisβ-amyrin synthase which can catalyse epoxy squalane intoβ-amyrin.3) Functional identification by expressed in G.StramineaPlant overexpression vector(pK7WG2D) and RNAi vector(pK7GWIWG) were constructed using gateway technique and transferred into 7d grown callus of Gentiana Straminea by particle gun.After 24hours dark cultivation,the calli was selected in the culture medium with 50mg/L kanamycin.Surviving calli were then transferred to differentiation medium(IB).Rooted seedlings were continuely selected by kanamycin in the differentiation medium.A total of 111 resistant lines(GsAS1overexpression plants 27 lines,GsAS2 overexpression plants 34 lines, GsAS1 RNAi plants 29 lines and GsAS2 RNAi plants 21 lines) were gained in this way.GsAS1 and GsAS2 specific PCR primers were designed to conform by amplify a fragment of GsAS1 and GsAS2 from genome of resistant plants.The transformation rate was caculated (GsAS1overexpression 2.86%,GsAS2 overexpression 3.13%,GsASIRNAi 4.00%and GsAS2 RNAi 2.94%).HPLC analysis determined that the G.Straminea overexpressed GsAS1 contents 17%-113%more oleanolic acid than the control line and the G.Straminea overexpressed GsAS2 contents 67%-146%more oleanolic acid than the control line.As expected the RNAi lines had lower oleanolic acid content than the control line It proves that over expression the GsAS1 and GsAS2 increased the accumulation of oleanolic acid in transgenic plants and GsAS2 made much greater contribution than GsAS1.4.Study on metabolic profiling of S.involucrate and S.lacosteiThe analysis of water-soluble substance in S.involucrate and S.lacostei showed that there were 29 volatile organic compounds(VOCs) in S.involucrate and 24 VOCs in S.lacostei.15 VOCs of them existed in both materials.What's more,a,a-dimethyl-benzenemethanol and acenaphthylene was first found in S.lacostei...