Over-expression and analysis of two endogenous UDP-glycosyltranferases genes in Korean wild ginseng (Panax ginseng C.A.Meyer)

Abstract

Panax ginseng C.A Meyer (Korean wild ginseng) is an ancient and medicinal plant that wildly used all over the world. The most beneficial compound involved inside of ginseng plants is ginsenoside, with the high attentions have been paid to ginseng researches, more and more ginsenosides had been extracted and identified from ginseng plants. Up to now, more than 70 ginsenosides have been reported from natural and processed ginseng. In the meantime, various functional genes which are engaged in ginsenosides biosynthesis pathway come to be known, including UDP-glycosyltransferases (UGTs). UGTs play roles in the final step of ginsenoside synthesis, after glycosylate reactions catalyzed by UGTs result in variety of ginsenosides. In order to explore the function of UGTs in ginenosides biosynthesis pathway and increase the content of ginsenosides in ginseng adventitious roots, two endogenous UGTs genes (PgUGT74AE2 and PgUGT94Q2) were transformed to Korean wild ginseng by Agrobacterium-mediated transformation. The adventitious roots induction procedure was optimized by applying different rooting hormones, and induced adventitious roots directly from callus. Because different phenotypes were observed in different hormone rooting medium during culturing, morphological and histological analysis were applied for recording. In case of hormones and different resources give rise to the concentration of ginsenoside, high performance liquid chromatography (HPLC) was supposed for ginsenoside contents analysis for both wild-type and transgenic lines. Callus derived and adventitious roots derived roots rooting by IBA was observed increasing of total content of main ginsenosides (Re, Rg1, Rc, Rb1, Rb2, Rd), especially ginsenoside Rb2. As callus acting as the most suitable host for plant genetic engineering, transformed callus derived roots rooting by IBA was supposed to produce transgenic roots in brief period. PgUGT74AE2 and PgUGT94Q2 transgenic lines generated within 6 month, including callus induction, transformation, roots induction from transformed callus. Transgenic candidates of PgUGT74AE2 and PgUGT94Q2 were selected by Immuno strip test (Agrastrip GMO, LL test Strips) as first confirmation. 9 lines of PgUGT74AE2 (74 L2, 74 L4, 74 3L2, 74 L2-2, 74 T2, 74 T3, 74 T5, 74 T6, 74 T7) and 7 lines of PgUGT94Q2 (94 L2, 94 T2, 94 T4, 94 T5, 94 T8, 94 T11, 94 T12) were screened from transgenic candidates. Total RNA was extracted from all transgenic lines, and cDNA synthesized from total RNA, cDNA used as template for RT-PCR analysis and bar gene confirmation. Genomic DNA extracted from all transgenic lines to performed southern blotting to analyze T-DNA region insertion copies. Incorporation with HPLC results of transgenic lines, PgUGT74AE2 transgenic lines have 4 lines (74 L2-2, 74 L2, 74 L4, 74 T2) and PgUGT94Q2 transgenic lines have 4 lines (94 L2, 94 T4, 94 T2, 94 T5) gained ginsenoside contents increasing compared with wild-type ginseng. And one unknown compound among these 8 transgenic lines showed content enhancement. Further analysis of unknown compound structure is in the management.