Functionanl Analysis Of DELLA Family Proteins In Peanut Stress Resistance

DELLA proteins are key negative regulators of gibberellin signaling pathway,which act as a bridge that connects the environmental signal to plant growth and development.According to the relevant research,gibberellin synthesis can be inhibited under multiple stress conditions,while DELLA protein is relatively stable,leading to plant growth inhibition,and increase plant tolerance to stress.The expression analysis of DELLA family genes in Arabidopsis thaliana showed that the expression of five DELLA genes in Arabidopsis thaliana was not obviously affected by cold,heat or hypoxic conditions.Different from Arabidopsis thaliana,peanut DELLA family consists of four members,and our previous studies have shown that AhDELLA3 a and AhDELLA3 b genes were strongly induced by drought and high salt stresses.In this study,we investigated the function of peanut AhDELLA family genes under drought or high salt stresses used the transgenic plants.We cloned the AhDELLA1 promoter and analyzed its expression activity in different plant organs or under abiotic stresses.In order to find the critical cis-elements on the inducible AhDELLA3 a promoter,the promoter was studied using the transgenic tobacco.These results provided useful information for further studies on the roles of AhDELLA in peanut stress resistance.The main results of this study were summarized as follows:(1)Stress resistance testing of the AhDELLAs transgenic tobacco plants.In our previous studies,transgenic tobacco plants of peanut AhDELLA1,AhDELLA2 and AhDELLA3 a were obtained.The transgenic plants were treated with drought and salt stresses at seedling stage to observe the stress resistance.The results showed that the growth of AhDELLA transgenic tobacco were all better than that of wild type tobacco after stress treatment.The activities of SOD and POD in transgenic tobaccos and wild tobacco were increased with the prolong time of treatmen.The SOD or POD activities of transgenic tobaccos were higher than that of wild type tobaccos.The germination rate of seeds was also observed on salt containing medium.The results showed that the germination rate of AhDELLA transgenic tobaccos were significantly higher than that of wild type tobaccos on 200 mM NaCl medium.These results demonstrated that AhDELLA family genes may play important roles in peanut resistance to drought and high salinity stresses.(2)AhDELLA1 and Ah DELLA3 a genes were transformed into peanuts by calyx tube injection.Expression vectors pCAMBIA2300-AhDELLA1 and pCAMBIA2300-AhDELLA3 a were transformed to Agrobacterium tumefaciens c58c1.The flower of the peanut Luhua 14 was injected with the Agrobacterium cultures.The peanut seeds were harvested and the transformation efficiency of pCAMBIA2300-AhDELLA1 was about 7%,and the transformation efficiency of pCAMBIA2300-AhDELLA3 a was about 6%.(3)AhDELLA1 promoter sequence was cloned and the GUS expression vector was constructed.The tobacco leaves were transformed by Agrobacterium LBA4404 which contained the vector pCAMBIA1381z-AhDELLA1pro::GUS,and cultured for 2-3 days in dark.The expression of GUS was detected in all organs of the transgenic plants,including leaves,roots,stems,flowers and immature seeds.GUS expression of transgenic tobaccos was significantly enhanced after salt stress treatment.The expression of GUS was mainly in leaves,while weak signal was detected in hypocotyls.Histochemical staining results showed that AhDELLA1 promoter was responsive to salt stress.(4)Identification of key regulatory elements in AhDELLA3 a promoter.It was proved that the expression of AhDELLA3 a was promoted under diverse stress conditions.To screen the upstream regulatory factors,we first analyzed the promoter of AhDELLA3 a.AhDELLA3a promoter contained multiple stress-induced elements.In order to further understand the function of these cis-acting elements,different promoter deletion fragments were designed to construct expression vectors together with the reporter gene GUS.Different length of AhDELLA3 a promoter was amplified using PCR,and constructs that GUS driven by these promoter fragments were constructed.Tobacco was transformed by Agrobacterium,and then GUS expression was observed the transgenic tobaccos.