Cloning And Functional Analysis Of ZmCPN3 Gene In Maize

Copines gene was originally found in paramecium,subsequent studies found many homologous Copines genes widely exist in mammals,nematodes and Arabidopsis thaliana.However,we know little about the Copine family in plant.Studies have shown that AtCopine1 play important roles in regulating plant disease resistance.Through bioinformatics analysis,maize contains the higher homology gene ZmCPN3 with AtCPN.To explore the functions of copine proteins in the process of maize diease resistance,we cloned the full-length of ZmCPN3 gene and studied the subcellular localization,relationship between ZmCPN3 gene expression and free salicylic acid content,disease resistance of transgenic plants to rice sheath blight and stripe fungus.The main results were as follows:1.We cloned the full length cDNA of ZmCPN3 gene from maize,sequence analysis showed the length of CDS is 1161 bp,containing 11 introns and 12 exons,encoding 386 amino acids.Transfered 35s-CPN3-GFP fusion vector into root cells of rice.GFP signal was detected under a fluorescence microscope and the result is: ZmCPN3 located on the cell membrane.2.Tissue expression pattern analysis of ZmCPN3 gene,ZmCPN3 gene expressed highest in stem and about 28 times in root;Expression quantity in leaf about 2 times of root,in ear the expression quantity is 3 times of the root.3.ZmCPN3 overexpression vector was constructed.In the experiment obtained eight independent transformation event through agrobacterium-mediated transformation of rice.Determination of free SA content of T1 generation plants and CPN3 overexpression plants have a slightly high than CK.Expression pattern analysis of ZmCPN3 gene in transgenic plants under salicylic acid treatment.ZmCPN3 expression content rapidly rised more than twice than CK after 1 hour,then dropped to a lower level 3 hours later.4.CK and ZmCPN3 overexpression transgenic rice inoculated with rice sheath blight and bacterial stripe at sword leaf stage.After inoculation with bacterial stripe,transgenic plants showed serious disease compared with CK and the wither appeared in whole leaves.In addition,for sheath blight,incidence of transgenic plants focused on 25%~50% and disease progression is 5 while wild type incidence is 5%~25%,the disease progression is 3.The results proved ZmCPN3 in plant negatively regulate on plant disease resistance.5.ZmCPN3 overexpression plants and CK were subjected to drought stress.transgenic plants showed severe leaf rolling and wilting,whereas CK plants exhibited such phenotypes to only a slightly extent after drought treatment.Identification the drought resistance of rice,survival rate of CK was 87%,However,transgenic plants were 31%,36%.Illustrating ZmCPN3 gene negatively regulate drought resistance of plants.