Construction Of RNAi Vector Of Cycloartenol Synthase(CAS) Gene And Genetic Transformation In Betula Platyphylla Suk.

Triterpenes in Betula Platyphlla suk.have vital activities of anti-HIV and anti-tumor,and the active components of white birch triterpenes,(betulin,oleanolic acid,damareneglycol)and the sterol came from 2,3-oxidesqualene,the common precursor in MVA pathway.The precursor would be catalyzed by various oxidized squalene cyclases(OSCs),to synthesize different triterpenoids and sterols.Based on the metabolic pathway of triterpenes in white birch,we intend to clone the gene of cycloartenol synthase(BPX)and to perform the bioinformatics analysis.Meanwhile RNAi expression vectors of CAS gene being constructed by different methodswere transforming into white birch seedlings.The synthesis and accumulation of triterpenoids component and the changes of the key enzyme genes expression in triterpenoids synthesizing pathway were investigated after the suppression of CAS gene,so as to clarify the effect of blocking the branch of steroids synthesis in both the synthesis of betulinic acid and oleanolic acid and the regulation of CAS gene in the network of triterpene metabolism.This study will lay the foundation for the genetic modification and biotechnology utilities in the triterpenoids metabolism pathway in white birch.The results of this study were as follows:1.The full-length sequence of CAS cDNA in Betula Platyphlla suk.,tentati'vely named BPX3,were obtained by RT-PCR.Molecular characteristics,physical and chemical properties,homology and phylogenetic tree of BPX3 were analyzed by bioinformatic softwares.The result showed that the full-Iength cDNA of BPX3 ORF(open reading frame)was 2262 bp,encoding 735 amino acids.There was a specific conserved sequence,ISOPREN_C2_like superfamily(in 97?736th position),encoding a hydrophilic protein with a helix,extended chain,random coil and four secondary structures of transmembrane helix.The homology among BPX3,CAS genes of Betulaplatyphylla var.japonica,Azadirachta indica and other species reached 78?96%.It revealed that BPX3 protein had catalytic activity,isomerase activity,intramolecular transferase activity and cycloartenol synthase activity for molecular function.And this protein participated in the isopentenyl pyrophosphate biosynthesis process and methylerythritol 4-phosphate pathway,chlorophyll biosynthesis,carotenoid biosynthesis,thylakoid membrane,organization,pollen development,unsaturated fatty acid biosynthetic process,pentacyclic triterpenoid biosynthetic process,sterol biosynthetic process,phosphatidylglycerol biosynthetic process and tetracyclic triterpenoid biosynthetic,process.The protein was the cellular component of integrating to the membrane protein and in vacuole.2.The distribution and content of oleanolic acid and betulin in each organs of the four-year,six-year and eight-year white birch were detected.Results showed that oleanolic acid and betulinexisting in the leaves,stems and root barks of the birches with different ages mainly concentrated in the stem barks while low content in leaves and root barks.The content of oleanolic acid and betulin in four-year white birch had reached a highest accumulation in the stem barks.Genes expression by qRT-PCR were analysized,showed BPX1(cycloartenol synthase genel)and BPX2(cycloartenol synthase gene).in leaves in the growing season had the highest expression in late September,while lower expression levels of BPW(oleanolic acid gene)and BPY(betulin gene).And two peaks of BPY expression in stem bark appeared in late July and September.The expressions of other three OSCs genes were less than BPY,whereas the BPX2 and BPW expression levels increased gradually,from the end of June to late September.BPY in the roots hadhigh expression levels in late July and late October.BPW had the highest expression in August,followed by the end of September and the late October.BPX peaked at late August with lower expression levels overall.These results indicated that the accumulation and distribution of the genes expression of triterpenoids were similar and regulated by the seasons significantly.3.A New RNAi plant transformation vector,ihpRNA was modified from pRNAi-GG,supported by Dr.Yan based on full-length gene sequences of BPX1,BPX2 and BPX3.According to the requirements for RNAi and principles of the vector pRNAi-GCA difFerent fragments of CAS gene were selectedfor designing primers.Then the interference fragments vectors,BPX1-A(N1),BPX1-B(N2)and BPX123-C(interfering both BPX1 and BPX2,N3)were successfully constructed into the vector.Fragment(N4)interfering the homologous region of BPX2 and BPX3 were construct into the vector and named p121-N4 using chemical synthesis.Using tri-parental mating.BPX1-A,BPX1-B,BPX123-C and BPX23-D were transferred into Agrobacterium tumefaciens successfully from E.coli.Four transformants of engineering strains were obtained which can be used for genetic transformation and for producing CAS gene RNAi transgenic white birch.4.The RNAi vectors contained CAS gene,p121-N4,were transferred by Agrobacterium tumefaciens-medidXed genetic transformation into clones of white birch,using the leaf-disc transformation method.Then aepuired four transgenic white birch seedlings from 35 transformants wereverified by PCR and Southern bloting with NptII.qRT-PCR analysis showed that the gene expression of BPX2 were suppressed in two seedlings(Y-12,B-6)with 3%and 48%of the wild-type gene expression.It was also found that the expression of BPX2 in transgenic white birch seedlings Y-12 and B-6 were inhibited,while the relative expression BPY and BPW amount significantly increased.And in Y-12,relative expression of BPY and BPW were 2.95-fold and 2.89-fold of the control.In B-6 transgenic plants,other genes were upregulated except BPX2,While BPY and BPW expression levels increased to 70.9 times and 84.10 times of the wild-type.It was also found that the content of oleanolic acid and betulinic acid in callus of Y-12 and B-6 were significantly higher than the wild type with 5.97 times and 8.92 times detected by HPLC respectivly.B-6 were increased significantly with 2.10 times of the control.These results indicateed that RNAiof BPX2 in white birch reduced the BPX2 expression levels significantly,while the expression of BPW and BPY genes increased significantly,subsequently it significantly promoted 2,3-oxidation squalene,the precoursor of triterpenoids to the synthesis and accumulate oleanolic acid and betulinic acid which were the target products of triterpenoids by the downstream pathway.