Cloning Of CsLEA Gene From Cleistogenes Songorica And Transformation Of Arabidopsis

Drought, salinity and low temperature are the most important of several stress factors limit plant growth and crop yield of many abiological adversities. Hydropenia can damage to plants, even induce foliage to death. They also might reduce crop yield and restrict crop planting area. LEA protein(Late Embryogenesis Abundant Protein)has a special structure as the dehydrating protecting agent, and LEA could take part in osmotic regulation as a regulatory protein and molecule chaperone. Accumulation of LEA protein avoided damage of biomacromolecules, especially membrane system in some extent.In this study, cleistogenes songorica CsLEA gene was isolated from c.songorica using hiTAIL-PCR and RT-PCR. Then the CsLEA gene was analyzed using bioinformatics method. We constructed three plant expression vectors using Gateway and other technique, then, pBI12135S::CsLEA and pBI121rd29A::CsLEA were transformed into inflorescence of Arabidopsis thaliana plants by agrobacterium-mediated method.The main results were summarized as follows:1. Cloning of LEA Gene and its PromoterA CsLEA gene and its upstream regulation sequence were isolated from C. songorica. The gene was817bp in cDNA sequence, coded a clone a protein product of153animo acides. The protein about36385.6Da, and without any transmembrane region. The length of CsLEA gene5’end and its upstream were972bp.2. Construction of plant expression vectorConstructed three vectors:pBI12135S::CsLEA, pBI121rd29A::CsLEA and pEarlyGate101bar-CsLEA.3. Genetic transformation and molecular biologyThe CsLEA gene which were regulated by promoter rd29A and35S promoter respectively, were transferred into Arabidopsis by agrobacterium-mediated method. Obtained three resistant plants of pBI12135S::CsLEA and thirteen resistant plants of pBI121rd29A::CsLEA.4. Evaluated the resistance of transgenic ArabidopsisExperiment in the stress resistance of T2generation resistant plants. The MS medium experiments showed that, when the NaCl concentration for the50mM, the wild-type plant growth was significantly suppressed, while the growth of transformed plants, the concentration reached100mM, the growth of wild-type and transformed plants were inhibited; the wild-type plant growth was suppressed when sorbitol concentration as300mM, while transformed plants grow well when the concentration reaches400mM, the growth of wild-type and transformed plants were inhibited. A pot experiment showed that, in droμght stress Arabidopsis transgenic plants growth conditions was significantly better with the wild-type plants.