Specific promoters for genetic engineering of rubber tree

Cloning of ethylene-inducible and/or laticifer-specific promotors from genes expressed in rubber tree latex was untertaken with the objective to optimize transgene expression in genetically engineered rubber tree. Glutamine synthetase (GS) and hevein (hev) gene promotors were sought for, based on the fact that (1) overexpression of the gs genes was observed after ethylene treatment in latex, and (2) the hevein protein has been found in the laticifers only. Several genomic clones were obtained. Partial sequencing revealed that both hevein and gs are encoded by multigene families. Hevein genes could be classed in two groups, based on sequence homology. Three different gs genes (gs1, gs2 and gs3) were identified. Gene expression analysis revealed that: (1) both gs1 and gs2/gs3 (without possible distinstion) were responsive to ethylene in latex, with gs1 apparently strictly induced and gs2/gs3 overexpressed; (2) the very high expression level of the hev gene in latex masked any potential effect of ethylene; (3) gs1 and gs2/gs3 were differentially expressed in tissues from in vitro culture at various stages of development; (4) both gs and hev genes were expressed in callus; (5) hv gene expression increased concomitantly with the development of differentiated laticifers. Two different hevein promoters (hev1 and hev4) and two gs promoters (gs2 and gs3) were isolated and cloned in fusion with the gus reporter gene in a transformation vector. The different strategies envisaged to analyse the functionality of the isolated promoters are presented. The choice of a promoter, for a genetic engineering programme, needs to take into account both the physiology of the plant and the application being sought, in order to optimize the expression of the transgene. In the case of rubber tree (Hevea brasiliensis), genetic engineering is being considered in order to improve existing varieties, but also as a physiological study tool intended to analyse or confirm the function of certain genes. In addition, several teams are interested in Hevea as a "green bioreactor" for production by the latex of marketable molecules other than rubber (molecular farming). In the case of an application seeking to improve rubber production or for molecular farming, it is important that the promoter used guarantee optimum expression in the latex cells (laticifers), and if possible in the latex cells only, so as to disturb the tree as a whole as little as possible. Hence our search for a promoter specific to those tissues. We are also seeking for a promoter that can be induced by ethylene, in order to induce or strengthen transgene expression under farming practices calling for ethylene treatment (ethephon or ethylene gas) to stimulate production. The use of ethylene stimulants needs to be limited, since over-stimulation, like over-tapping, can cause a physiological imbalance in the tree and penalize production. One possible strategy to limit this risk is to genetically modify Hevea in such a way that certain endogenous genes involved in protecting cells from oxidative stress, which is itself generated by overexploitation, are over-expressed. By using a promoter that is inducible or over-expressed by ethylene, the activity of the transgene would be stimulated when most needed by the plant. (Résumé d'auteur