Marker gene excision in transgenic Brassica napus via Agrobacterium-mediated Cre/lox transient expression system

Abstract

Oilseed rape (Brassica napus L.) is one of the most important oil crops in the world. However, study on marker-free transgene of B. napus for bio-safety purpose is limited in this allotetraploid crop. In order to advance marker gene excision research, a newly designed Cre/lox system combining crossing and auto-excision strategy was introduced into B. napus. The system consists of 2 sets of independent vectors including pC35Spro::T7RP carrying T7 RNA polymerase and pCT7pro::Cre carrying T7 promoter respectively. After hybridization of 2 according types of transgenic B. napus, marker gene would be removed as T7 RNA polymerase facilitate T7 promoter to promote Cre gene expression. Totally 52 and 46 positive T0 transgenic lines of these 2 vectors were obtained after identification by PCR and test trip. T1 plants from 3 T0 positive pC35Spro::T7RP lines and 2 T0 positive pC35Spro::T7RP lines were identified as single copy according to segregation ratio and were chosen for crossing. However, expression of CP4 EPSPS (glyphosate resistance gene) and OXY (bromoxynil resistance gene) were not found in F1 progeny, which proved that the excision was not complete. The possible reasons for our limited success were investigated and detailed analyses were performed. Although this system is not applicable for generating transgenic B. napus free from selectable marker gene, it provided valuable experience and clue for further improvement of this technique. Many other advantages and further improvement will be progressed in future work.