Functional Study Of Cyclophilin And Calcium Signal In Response To Salt Stress In Peanut

Peanut(Arachis hypogasa)is an important oil crop in China.With the aggravation of soil salinization,the available tillage area in China is decreasing constantly.In order not to compete with grain,planting peanuts in saline-alkali land has become a new trend of agricultural development.However,salt stress in saline soil usually causes ion toxicity,imbalance of osmotic pressure and oxidative damage to plants,which poses a great threat to high yield and good quality of peanut.Root system is the first part of plant to feel salt stress.Whether the root development is good or not during peanut germination directly affects the growth and yield of peanut in the later stage.Therefore,the study on the mechanism of salt tolerance of peanut roots during germination is of important reference significance for the cultivation of peanut in saline soil.Plant cyclophilin is a polygenic,highly conserved protein family and it has the activity of peptidyl prolyl cis-trans-isomerase.A large number of studies have shown that cyclophilin plays an important role in the response of many plants to abiotic stresses,such as high salt,drought,heat,cold and high light.But so far,there is no research on AhCYP in salt tolerance.In this study,the full-length sequence of AhCYP was obtained from the root system of "Huayu 22" peanut during germination,and the expression vector was constructed,then the method of Agrobacterium-mediated transformation was used to transfer the AhCYP into the wild-type tobacco,and the salt tolerance of transgenic tobacco was evaluated.In addition,this study applied 5 mM EGTA,0.05 mM CPZ and 2 mM LaCl3 to peanut roots during germination under 150 mM NaCl stress.Then,the role of calcium signal in the response of peanut roots to salt stress during germination was studied.The main results are as follows:1.Functional Analysis of AhCYP under Salt stressThe gene sequence of AhCYP was amplified from the cDNA of "Huayu 22".The open reading frame of the gene is 519 bp,which can encode 172 amino acids.AhCYP belongs to hydrophilic protein,it has no signal peptide and no transmembrane domain,and it has 17 phosphorylation sites.The secondary structure of AhCYP is mainly irregular curl and alpha helix.In addition,the regulatory region of AhCYP promoter contains elements related to hormone response,light response,plant stress and defense gene activation.Phylogenetic analysis showed that AhCYP was closely related to cotton GhCYP1.Subcellular localization studies showed that AhCYP was localized in the cytoplasm.qRT-PCR was used to analyze the expression pattern of AhCYP in peanut roots during germination under stress,The results showed that AhCYP was up-regulated under salt stress and the expression level was the highest at 3 h.The expression of AhCYP was up-regulated at the initial stage of osmotic stress and oxidative stress,and then down-regulated.The above results indicated that AhCYP has different response mechanisms to different abiotic stresses.Analysis of transgenic AhCYP tobacco showed that transgenic tobacco plants grew well and had more seeds under normal growth conditions.Phenotypic experiments showed that transgenic tobacco showed resistance to NaCl,Mannitol and MV.Under salt stress,the germination rate,root activity,osmotic regulatory substance content and antioxidant enzyme activities of SOD and CAT in transgenic tobacco were significantly higher than those in wild-type tobacco,while the relative conductivity,MDA content and ROS accumulation were significantly lower than those in wild-type tobacco.According to these results,overexpression of AhCYP can improve the salt tolerance of transgenic tobacco.2.Functional study of calcium signal in response to salt stress in peanut root during germination.Salt stress had a serious inhibitory effect on the root system of peanut during germination,mainly in the aspects of inhibiting the main root elongation and lateral root growth,reducing root activity,increasing ROS accumulation,aggravating membrane lipid peroxidation,and reducing the activity of antioxidant enzymes.However,when calcium signal inhibitor was applied after salt treatment,the root system of peanut was short in shape and browned during germination.Compared with single salt stress,the activity of antioxidant enzymes in peanut root decreased during germination,which resulted in excessive accumulation of ROS in root and oxidative damage of root membrane.Calcium signal inhibitors inhibited antioxidant enzyme activity,which indicated that calcium signaling pathway might play a role in the response to salt stress through regulating the activity of antioxidant enzymes.Calcium signal inhibitors inhibited the expression of AhCaM,AhCBLl,AhCDPK and AhCNGC15.At the same time,it also inhibited the expression of AhNHX3,AhNHX8,AhCYP,AhARF2,AhARF9,which was related to salt stress,and further speculated that calcium signaling pathway might respond to salt stress by regulating the expression of stress-related genes.In addition,calcium signal inhibitors inhibited the expression of ABA synthesis-related gene AhAO1,AhABA2,AhNCED1 under salt stress,hindered the biosynthesis of ABA,and affected the role of ABA-dependent signaling pathway in the response to salt stress in peanut root during germination.This suggested that calcium signaling pathway and ABA signaling pathway might co-regulate the response of peanut root to salt stress during germination.