Molecular Cloning And Expression Analysis Of Squalene Synthase And Squalene Epoxidase Genes From Betula Platyphylla Suk

Triterpenes in Betula Platyphlla suk. have vital activities of anti-HIV and anti-tumor. The active components of white birch triterpenes (betulin, oleanolic acid, damareneglycol) and the sterol come from 2,3-oxidesqualene, which is the common precursor in MVA pathway. The two key enzyme that directly involve in the synthesis of 2,3-oxidesqualene is the squalene synthase (SS) and squalene epoxidase (SE), so the content and activity of SS and SE plays an important role in birch triterpenoids yield. In this study, birch (Betula Platyphlla suk.) triterpenoid pathway key enzyme BpSS and BpSE two genes full-length cDNA and promoter of the genes were cloned, and were also investigated the expression patterns of genes BpSS and BpSE and enzyme activity.The results of this study were as follows:1. The full-length sequence of BpSS and BpSE cDNA in Betula Platyphlla suk. were obtained by RACE. Molecular characteristics, physical and chemical properties, homology and phylogenetic tree were analyzed by bioinformatic softwares. The results showed that the full-length cDNA of BpSS was 1588 bp, with an ORF of 1242 bp encoding 413 amino acids. There was a specific conserved sequence, Isoprenoid_Biosyn_C1 like superfamily, a squal_synth sequence and a SQS_PSY sequence. The amino acid sequence of BpSS shared 88% homology with that of Glycine max, Camellia oleifera. The BpSS was included in a separate clade consisting of members of the Theobroma cacao family to which BpSS belongs. The full-length cDNA of BpSE was 2040 bp, with an ORF of 1581 bp encoding 526 amino acids. Conserved domains (NCBI) that predicted the BpSE-deduced polypeptide sequence contained a SDR superfamily. The BpSE was included in a separate clade that consisted of members of the Withania somnifera family.2. The expression pattern of BpSS and BpSE genes were investigated under different phytohormone treatment at different time by using real-time RT-PCR. Results indicated that BpSS and BpSE were expressed in a constitutive manner in the tissues of roots, stems, and leaves, with the highest expression levels which was found in leaves and the lowest expression levels which was found in the roots. The expression of BpSS and BpSE was induced by MeJA、ethephon and ABA at different degrees. However, the SA treatment did not affect the BpSS and BpSE transcripts notably. Using triparental hybridization mediated transient transformation, the BpSS fused GFP protein were transiently expressed in onion epidermal cell, and the green fluorescence were detected in the nucleus and cytoplasm of onion cell. The results revealed that the BpSS protein was targeted to cytoplasm.3. The promoter of BpSS and BpSE was isolated using TAIL-PCR approach, and analysis showed that the promoter is 965bp、1193bp in length and contains some phytohormone-response and stress-response cis-acting elements such as CGTCA-motif, GARE-motif, TGA-element, TC-rich repeats.4. Both of the BpSS and BpSE genes were inserted into the yeast expression vector pYES2 and recombinant plasmid (pYES2-BpSS andpYES2-BpSE) and pYES2 were transformed into Saccharomyces cerevisiae INVScl to characterize the synthesized product and enzyme activity. The transformants were cultured for 12 h, the squalene content of galactoseinduced BpSS expression yeast cells was 13.2 times of control (empty vector control yeast cells) by high-performance liquid chromatography (HPLC) test method. And, the squalene epoxidase activity of induced BpSE expression yeast cell was about 11.8 times of control. These indicated that we cloned birch BpSS and BpSE that were indeed involved in the synthesis of triteropenoids.