Expression analysis of the recombinant Catharanthus roseus deacetylvindoline 4-O-acetyl transferase in tobacco plants

Bui Thi Ha, Nguyen Thi Ngoc Lan, Nguyen Thi Tam, Le Van Son, Chu Hoang Mau
2018 Australian Journal of Crop Science  
Catharanthus roseus (L.) G. Don contains about 130 types of alkaloids, including vincristine and vinblastine, which are outstanding drugs for cancer. The C. roseus deacetylvindoline-4-O-acetyl transferase (CrDAT) is a key enzyme which catalyzes the second to the final reactions in the vindoline way. The low content of indole alkaloid in C. roseus plants and the high cost of indole alkaloid production have promted many research to improve indole alkaloid yield in this plant. The aim of this work
more » ... was to express recombinant CrDAT in tobacco, a model plant, to create the basis for the overexpression of the gene encoding CrDAT (GenBank LN809930) in C. Roseus plants. In this study, the 35S-DAT-cmyc structure was transferred to tobacco and the transgenic tobacco lines was generated. The T1 generation was then analyzed by Western blot method and ELISA analysis. Southern blot assays confirmed that the CrDAT gene was completely introduced into tobacco genome by Agrobacterium-mediated transformation. The recombinant CrDAT protein of 51.5 kDa in size was successfully expressed at the seven transgenic tobacco lines. The recombinant CrDAT protein content of transgenic tobacco lines were 2.75 -5.35 (g. mg -1 of total protein) range and the recombinant CrDAT protein content of the T0-1 line was highest (5.35 g. mg -1 of total protein). MIAs_monoterpenoid indole alkaloids; D4H_deacetoxyvindoline 4-hydroxylase; rCrDAT_recombinant CrDAT; T0, T1_generations of transgenic plants; TDC _tryptophan decarboxylase; WT_ the wild-type tobacco plants (non transgenic plant).
doi:10.21475/ajcs.18.12.07.pne1077 fatcat:qx3mlodyhjhnbgbpuzhe7apnsm