Cloning and Expression Analysis of 4 Oxidosqualene Cyclase Genes in Tripterygium wilfordii
ZHU Chuan-Shu1,2*, LIU Yan1, PU Shi1, HUO Yan-Bo1, ZHANG Bin1, FENG Jun-Tao1,2, ZHANG Xing1,2
1 College of Plant Protection, Northwest A&F University, Yangling 712100, China; 2 Biopesticide Technology and Engineering Center of Shaanxi Province, Yangling 712100, China
Abstract：Tripterygium wilfordii is an important medicinal plant that has anti-cancer, anti-inflammatory and immune system regulation effect, and also has strong insecticidal activity. Celastrol is the most representative triterpenoid in T. wilfordii which has high pharmacological activity for medical application and potential development value. Oxidosqualene cyclase (OSC) is considered to be the key rate-limiting enzyme in the triterpenoid biosynthesis pathway. The present study cloned 4 OSC genes, TwOSC1 (GenBank No. MH412928), TwOSC2 (GenBank No. MH412929), TwOSC3 (GenBank No. MH412930) and TwOSC4 (GenBank No. MH412931), from T. wilfordii. The ORFs of TwOSC1, TwOSC2 and TwOSC3 were all 2 289 bp and encoded 762 amino acids. The TwOSC4 ORF was 2 280 bp and encoded 759 amino acids. Bioinformatics analysis showed that the amino acid sequence similarity of the 4 TwOSCs was high, and the similarity of multiple sequence alignments between each 2 sequences was 53% to 77%. Phylogenetic analysis showed that TwOSC1 clustered with β-amyrin synthase, TwOSC2 and TwOSC4 clustered with cycloartenol synthase, and TwOSC3 clustered with lupeol synthase. The expression patterns of 4 TwOSC genes in different tissues and the expression level in T. wilfordii hairy roots treated by methyl jasmonate (MeJA) were detected by qRT-PCR. TwOSC1 had a very strong tissue-specificity and was only expressed in the root. However, TwOSC2, TwOSC3, and TwOSC4 were expressed in all tissues. The expression level of TwOSC1, TwOSC2 and TwOSC4 increased by different degrees in T. wilfordii hairy roots after MeJA treatment. But MeJA treatment inhibited the expression of TwOSC3. The content of celastrol that was detected by high-performance liquid chromatography (HPLC) increased after MeJA treatment in T. wilfordii hairy roots. The change of TwOSC1 expression was consistent with the change of celastrol content after MeJA treatment. Above results indicate that TwOSC1 may be involved in the synthesis of celastrol in T. wilfordii hairy roots. The present study provides some basic data for the synthesis and regulation of triterpenoid in T. wilfordii.
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