Construction Of SSH Libraries And Molecular Cloning, Expression Of Genes Involved In Saponins Biosynthesis Of Panax Notoginseng

Panax notoginsen (Burk.) F.H.Chen, a well-known medicinal plant, is indigenousin Yunnan province. One of the most important bioactive compounds of the plant istriterpene saponins which has a wide range of therapeutic effects and pharmacologcalactivity on hematological system, nervous system, immunological system, substancemetabolism, anti-fatigue, cardiovascular diseases, and anti-cancer, anti-inflammatory,anti-aging. Percentage content of saponin in Panax notoginsen tissues is related tothe growth phase and different parts of the plant in which root is the most importantpharmacological activity part. Moreover, the percentage content of saponin in1-year-old root is much lower than that in 3-year-old root. It was assumed that thedifferent content of saponin was the result of differential expression of the genesinvolved in saponin biosynthes. Therefore, it was very important to clone these genesand investigate their functions. Forward and reverse suppressive subtractivehybridization (SSH) libraries were constructed by using mRNA prepared from3-year-old root and 1-year-old root. The Forward library (3-year-old root library) had2000 clones and reverse library (1-year-old root library) had 500 clones.Therecombination efficiency of the both subtraction libraries with the inserted fragmentsbetween 200 bp and 900 bp was 95%. The efficiency of suppressive subtractivehybridization was 32,000 fold. All these results indicated that both SSH librarieswere qualified for further studies.Plasmids with 500 bp inserted fragment were selected from each of the twolibraries for dot blotting hybridization in which DIG was used to label probes from3-year-old and 1-year-old SSH PCR products, respectively. A total of 110 genes were found to be only or highly expressed in 3-year-old root and 80 genes only orhighly expressed in 1-year old root. To confirm further the differential expressions ofthe genes, 6 genes from the 3-year -old roo(?) were chosen for RT-PCR analysis. Theresults indicated that dynamin GTPase effector, gamma-glutamyl hydrolase, multi-copper oxidase typeâ… family protein, IspG protein, NADH oxidase family proteinwere up-regulated, whereas, S-adenosyl-L-homocysteine hydrolase gene wasdown-regulated. Comparison of homologies of 6 differential expression genes infunction analysis suggested that IspG was an key gene in dammarane-type saponinsbiosynthesis parthway and the other 5 were directly or indirectly involved indammarane-type saponins biosynthesis.Bioinformatics analysis revealed that the nucleotide sequence of the IspG ESTcontained 524 nucleotides, encoded 146 amino acid residues with 15.636 KDa. Thegene had 90% identity with that from the other plants and 50% identity with thatfrom microorganisms. There were a domain related to transcription andN-myristoylation site, Casein kinaseâ…¡phosphorylation site, N-giycosylation Site,Protein KinaseC phosphorylation Site in IspG protein located in cytoplasm.A total of 91 clones from both cDNA libraries were sequenced and the sequenceswere assembled. The results indicated that there were 4 contig, 83 singlet, 87 unigene.There were 23 cDNA clones with poly A, which were accounted for 26.4% of tatolclones. Function analysis showed that function-known genes were 58 accounted for66.6% of tatol genes and classified into 11 categories. Most of them related tometabolism and secretory pathway. Function-unkown genes were 29 genes andaccounted for 33.3%.Thirty-seven clones with reading through amino acid sequences were analyzedby bioinformatics analysis. Signal peptide and membrane-spanning regionsprediction revealed that 3 clones had membrane-spanning regions and 8 clones weresignal protein or signal anchoring proteins. Functional sites prediction suggested thatthese genes were exocytosis and regulated cell aging and cell cycle. Functionalstructural domain prediction showed that there were 10 genes without functionalstructural domains and most genes were related to signal transduction, transcription regulation, electron transport. Subcellular localization analysis displayed that theproteins encoded by these genes were in cytoplasm.Squalene epoxidase (SE) was one of the rate-limiting enzymes in the triterpenesaponins biosynthetic pathway. One full-length cDNA encoding squalene epoxidasewas isolated from the root of Panax notoginseng by PCR according to the nucleotidesequence of squalene epoxidase gene of Panx ginseng. The squalene epoxidasecDNA (Accession no. DQ386734) contained 1622 nucleotides and encoded 537amino acid residues with 59.139 KDa and pI of 8.81. The protein had 98% identitywith that of Panax ginseng and different identities with those in other SE families.Panax notoginseng squalene epoxidase presented a FAD function domain,NAD(P)-binding Rossmann-fold domain, FAD/NAD(P)-binding domain,hydrophobicity, 4 transmembrane helices. All of these suggested that squaleneepoxidase might be a microsomal membrane-associated enzyme and would besecreted out of the cytosol with many of the catalytic properties and appearance inmany biological processes.Comparison showed that there was a WD domain, G-beta repeat in squaleneepoxidase of Panax notoginsen but not in that of Panax ginseng and this protein itthe two species was located in plasmid. Codon usage preference was not strong in thegene.The relative expression levels of the squalene epoxidase gene in various organsof Panax notoginseng were determined by quantitative real-time polymerase chainreaction. The results showed that the squalene epoxidase gene was expressed mainlyin roots, especially in 3-year-old roots. A very weak squalene epoxidase geneexpression was detected in one-year-old leaf and stem. These results suggested thatsqualene epoxidase gene expression was tissue-type specific and could be useful forforther study on the pharmacological activity.