Construction Of Expression Vector Based On Deoxyglucose Selection And The Application For Transformation Of CBF1 Gene In Lotus Corniculatus L.

Abiotic stress, especially low temperature, drought and high salt, 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 cold regulated genes, many results have been reported that those genes were transferred into the plants successfully and consequently improved the ability of tolerance against low temperature. Most of these genes are regulated by CBF transcription factors, so it is a novel channel to introduce the transcription factor genes into plants to improve crop cold resistance.In this study, the deoxyglucose selection system was firstly set up in crowtoe(Lotus corniculatus), and then the plant expression vectors carrying transcription factor CBF1 were constructed based on both herbicide and deoxyglucose selection systems, respectively. Transformation of CBF1 gene was performed by the Agrobacterium- mediated co-cultivation method. The main results are as follows.1. Obtainment of the genes: According to the DNA sequence of transcription factor CBF1 gene published in the GenBank, a pair of primer was designed and synthesized .Using the total genomic DNA of Arabidopsis thaliana seedling as templates, the full length of CBF1 gene was amplified by PCR and cloned into T-Easy vector. DNA sequencing result indicated that the target fragment showed 100% identity to the gene sequence in the GenBank. The selection gene DOGR1 was obtained by digestion of pBinAR::DOGR1 vector with restriction enzymes SalI and EcoRI.2. Construction of plant expression vectors: Three of plant expression vectors, including pCAMBIA3300-CBF1, pCAMBIA5300 and pCAMBIA5300-CBF1, are constructed. All the CBF1 and DOGR1 genes in those vectors are drove by the same promoter CaMV35S.3. Establishment of the deoxyglucose selection system in crowtoe: The system has been built through the screening of different 2-deoxyglucose concentration. We found that a small part of crowtoe cotyledons differentiated at first on the medium with 600 mg/L 2-deoxyglucose, but died gradually afterwards. When the concentration of 2-deoxyglucose is higher than 1000mg/L, no crowtoe cotyledons could be survived to differentiate.4. Obtainment of the transgenic crowtoe and its investigation: The rates of transformation based on both deoxyglucose selection and herbicide selection are similar. Under the normal growth condition, the proline contents in transgenic plants derived from both selection systems are litter higher than that of untransformed plants, and synchronous increased dramatically after 4℃treatment, reached the maximum value twelve hours later, respectively. These results demonstrated that over-expression of CBF1 gene could lead to increased proline contents in transformants and resulted in significantly improvement of cold tolerance of the transgenic plants.To our knowledge, it is the first time to set up the deoxyglucose selection system and the transformation using CBF1 gene in Lotus corniculatus. Our main aim is to investigate whether the expression of CBF1 gene from Arabidopsis could confer stress tolerance in Lotus corniculatus.