Search

Oilseed rape (Brassica napusL.) is one of the most important oil crops in the world. However, study on marker-free transgene of B. napusfor bio-safety purpose is limited in this allotetraploid crop. In order to advance marker gene excision research, a newly designed Cre/loxsystem 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 Cregene expression. Totally 52 and 46 positive T0transgenic lines of these 2 vectors were obtained after identification by PCR and test trip. T1plants from 3 T0positive pC35Spro::T7RP lines and 2 T0positive 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 F1progeny, 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. napusfree 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.

Bisphosphonates are inhibitors of bone resorption and are used increasingly as therapeutic agents for treating clinical disorders of skeletal metabolism. Their mode of action is still not fully understood. The demonstration that methylenebisphosphonate, a simple methylene analog of pyrophosphate, inhibits the axenic growth of amoebae of the slime mold Dictyostelium discoideumand is incorporated into adenine nucleotides suggested that this organism might be useful in elucidating the cellular effects of bisphosphonates. We examined 24 bisphosphonates, including all those of clinical interest as inhibitors of osteoclast‐mediated bone resorption in vivo, for their effects on D. discoideum.All the geminal bisphosphonates inhibited growth of Dictyostelium, although the effectiveness of individual compounds varied widely. When the bisphosphonates were ranked there was a remarkable similarity between the order of potency as inhibitors of growth of Dictyosteliumand the order of potency as inhibitors of bone resorption. Thus, bisphosphonates with more complex side‐chain structures, especially those containing a nitrogen group, were more potent than simple substituted bisphosphonates, some inhibiting Dictyosteliumgrowth even at concentrations below 10 μM. It therefore appears that the mechanism by which bisphosphonates prevent Dictyosteliumgrowth could be similar to the mechanism by which these compounds affect the activity of osteoclasts. Because the mechanisms of action of bisphosphonates on osteoclasts remains unclear, Dictyosteliummay provide an additional model for studying the biochemical mode of action of bisphosphonates. Furthermore, these studies suggest that Dictyosteliummay also be a convenient organism for rapid evaluation of potentially active bisphosphonates.