Attainment Of Transgenic Crowtoe (Lotus Corniculatus L.) Transformed With The Selective Marker-free Vector Harbouring Trehalose-6-phosphate Synthase (TPS1) Gene

Abiotic stress,especially low temperature,drought and high salinity,has been a negative factor in the agricultural activities over past years and become more serious due to the change of global climate. It is very important to improve the plant tolerance against various abiotic stresses by means of plant molecular technology in agriculture. With the discovery of drought-resistent genes,many results have been reported that those genes were transferred into plants successfully and consequently improved the ability of tolerance against drought.Selective marker gene is usually used to select transformed cells or tissue during gene transfer. However,it is useless once transgenic plants are produced,its existence might be harmful to environment and plant development. It will minimize the risk if the selectable marker genes were removed from the transgenic plants afterwards,and it is easier to be accepted for the consumer.More importantly,it is convenient for multiple genetic manipulations with genes of interests. In recent years, some transformation systems have been established to obtain marker-free transgenic plants.At present study,TPS1,a drought related gene coding trehalose-6-phosphate synthase,was isolated from yeast genome by PCR method and replace the GFP gene harboring pX6-GFP vector, forming the recombinant vector pX6-TPS1 with the function of Cre/loxP-mediated site-specific DNA recombination. The pX6-TPS1 vector was moved into crowtoe (Lotus corniculatus) via the Agrobacterium-mediated transformation. The antibiotic selective marker genes together with the Cre and XVE fragments were eliminated from the transformants under the inducement of 5μM/Lβ-estradiol to produce marker-free transgenic plants. PCR identification unveiled that TPS1 gene already integrated into the crowtoe genome. Lower rate of water loss from transgenic plants leaves showed that transformants have better water holding capacities than that of non-transformed plants. It hoped these results can promote the genetic transformation of other important crops in the future.