Variation Of Stress Tolerance Of Transgenic Arabidopsis Thaliana With ZmDGKs

PLC/DGK signaling pathway is a major part of the phosphoinsiol signaling pathway.It plays an important role in a variety of biological processes including growth,development and reponses to stress in plants.Diacylglycerol kinase(DGK) is a pivotal enzyme that phosphorylates diacylglycerol(DAG) to yield phosphatidic acid (PA).Both DAG and PA are signaling molecules in plant cells and are involved in kinds of signaling pathwanys.Therefore,DGK is thought to regulate the balance between DAG and PA through catalyzing their interconversion.Three DGK genes(ZmDGK1,ZmDGK2 and ZmDGK3) have been cloned from maize.They have different expression patterns in different tissues of maize analyzed by RT-PCR.Under the treatments of cold,drought and hormanes,they also showed specific expression patterns,which indicated they are possibly involved in vital processes.In this thesis,ZmDGKs were transformed into arabidopsis using Agrobacterium tumefaciens-mediated method,respectively.The transgenic plants were identified by PCR and RT-PCR analysis and homozygotic lines were obtained.To analyze the changes of AtDGKs in different stress conditions,we designed the primers for RT-PCR according to 3' or unconserved sequences of cDNAs of AtDGK gene family (AtDGK1～7).RT-PCR analysis of cDNA from five tissues of arabidopsis indicated that AtDGKs have different expression pattern in different tissues.Their expression under treatments of osmotic stress,salt,freezing and heat shock were analyzed by Real Time RT-PCR.Results showed that different individuals have different expression patterns in different conditions,which illuminated their different functions.We also analyzed the expression of AtDGKs and ZmDGKs in transgenic arabidopsis under the above treatments.Results showed that ZmDGKs had a high expression level in the plants,respectively,but the level was different from each other in different treatments.Just the opposite,the expression levels of AtDGK1 and AtDGK2 in the arabidopsis with over-expression of ZmDGK1 were reduced compared with wild type,especially in some treatments.AtDGK5 also showed lower expression in the arabidopsis with over-expression of ZmDGK2 and 3,respectively.These indicated that high expression of ZmDGKs might reduce the expression levels of some isforms of AtDGKs.Under the osmotic stress,over-expression of ZmDGK1 in transgenic arabidopsis seedlings displayed an improved tolerance compared with wild type.The transgenic seedlings showed higher germination rate of seeds,lower water loss rate and better phenotype than those of wild type.These results indicated the potential function of ZmDGK1 under the osmotic treatment.Meanwhile,over-expression of ZmDGK3 in transgenic arabidopsis also displayed better phentype and higher germination rate under the salt treatment,which illuminated that over-expression of ZmDGK3 in transgenic arabidopsis may play a positive role under the salt stress.In addition,the fusion expression vectors of ZmDGKs and gfp were constructed and transfomed to epidermal cells of onion by gene-gun.Results showed and ZmDGK1 located on the membranes in the cell,while ZmDGK2 and 3 located in the cytoplasm. We also cloned a new cDNA sequence named ZmDGK4 from maize in silico cloning combined with PCR,and the plant expression vector had been constructed and planned to transform arabidopsis or maize for further function analysis.