Functional Analysis Of Transcriptional Factor Genes Related To Stress And Obtaining Of Transgenic Lotus Corniculatus L.

Lotus corniculatus L. (Birdsfoot), a member of the perennial Leguminosae native to warm regions of Eurasia, is non-bloating for cattle and sheep that have the potential to become a major crop in temperate forage-producing regions of the world. The Lotus has a high nutritive proportion rank first in legume forages. It has a protein content of as high as 17.4% even after seed harvest. It also has low saponin content, palatability and strong regrowth habits advantages. At the same time, the Lotus as a pioneer grass is often used in establishing managed grasslands and artificial sowing. Leo, one of Lotus breeds introduced from Canada, is more tolerant to environmental extremes of cold, but less tolerant to salt and drought that influenced popularization in China. In order to solve the puzzle, we identify two highly resistant genes PeDREB2a and HhERF2 through functional analysis of the four transcriptional factor genes cloning from Populus euphratica and Halimodendron halodendron, respectively. PeDREB2a and HhERF2 are two factors with synogestic effects involved in plant resistance. Based on this fact, a series of plant expression vectors either harboring PeDREB2a or HhERF2 or both were constructed with non-resistance marker gene pmi and transferred into the Lotus genome to expect to improve salt endurance and drought resistance and adding fine varieties of herbage to Animal Husbandry Industry of China. The major work includes:1. By genetic recombination technology, the transcription factors, PeDREB2a, PeDREB2b, HhDREB2 and HhERF genes, cloned from Populus euphratica and Halimodendron halodendron, were amplified by PCR. Based on the vector pCAMBIA1302, two plant expression vectors rd29A::PeDREB2a and rd29A:: PeDREB2b were constructed under the controlling of rd29A promoter. (rd29A::HhDREB2 and rd29A::HhERF2 had already constructed by other person and conserved in our laboratory).2. The transcription factor genes were transferred into Arabidopsis. Under the selection of Hyg, 20,18, 23 and 27 pure transgenic lines of Arabidopsis were obtained for PeDREB2a, PeDREB2b, HhDREB2 and HhERF2 genes,respectively. These transgenic lines were further evaluated according to their germination rate, moisture content, soluble sugar and MDA of Arabidopsis induced by dehydration and high salt within germinating, seedling stage and leaf growth periods.The results thus obtained suggested that the introduction of these four genes into the Arabidopsis genomes can improve drought and salt resistance of the transgenic Arabidopsis at the different growth stages.3. The results further showed that the introduction of PeDREB2 and HhERF2 genes enhanced drought and salt tolerance of transgenic Arabidopsis significantly. Three plant expression vectors pmi-rd29A::PeDREB2a, pmi-rd29A::HhERF2, pmi-PeDREB2a::HhERF were further constructed basing on pCAMBIA1302-PMI, for transformation of the genes into Lotus corniculatus L.4. The constructed vectors were tranfered into the legume plant Lotus corniculatus"Leo"via Agrobacterium tumefaciens. PCR of the shoots thus obtained indicated that shoots had been transformed. Southern-blotting of selected regenerated plants further confirmed integration of the target genes into the Lotus genome. 7,12, and 5 transgenic lines of Lotus were obtained transfored by pmi-rd29A::PeDREB2a, pmi-rd29A::HhERF2, pmi-PeDREB2a::HhERF, respectively(Other transformed material detections are pending). Preliminary study of the transgenic regenerated plants under stress conditions showed that the transformed genes improved extreme environment tolerance of transgenic Lotus, especially the plant transformed with pCAMBIA1302-pmi-PeDREB2a::HhERF2.