Validation And Cloning Of Eggplant MiRm0002 Target Gene SmNTF3 And Preliminary Analysis Of Its Function In Response To Salt And Drought Stresses

Plants are often exposed to adverse environments during growth,such as extreme temperatures,high salinity,drought,and pathogen.These stresses are the main factors limiting plant geographical distribution and crop yield.MicroRNA(miRNA)is a class of non-coding small RNAs widely found in eukaryotes.It mainly plays a role in regulating plant growth and development,resistance to disease and secondary metabolism by regulating target genes.Therefore,target gene identification and functional analysis are the core content of studying miRNA biological functions.MiRm0002 is a new eggplant miRNA identified by high-throughput sequencing in our laboratory.Bioinformatics prediction indicates that TC9181 is the target gene of MiRm0002,and its matching region is in the 3’UTR region of the gene,which is not completely complementary.This gene encodes the mitogen-activated protein kinase SmNTF3.In this study,the target gene SmNTF3 was identified using degradome sequencing,qRT-PCR,and dual luciferase reporter gene detection method.Analysis of eggplant degradome sequencing data did not detect degradation fragment of SmNTF3,gene transcription expression analysis showed that the expression of miRm0002 and SmNTF3 did not show a completely negative correlation.These results indicate that if SmNTF3 is a miRm0002 target gene,the mode of action is not clipping,it may be translation inhibition.To test the hypothesis,this study analyzed the interaction between SmNTF3 and miRm0002 using a dual luciferase reporter gene detection system.The results showed that the luciferase activity of the SmNTF3-WT and m0002 mimic co-transfected tobacco group was lower than other groups,indicating that miRm0002 can recognize the SmNTF3-3’UTR binding site and inhibites the expression of SmNTF3 to reduce the enzyme activity.This result proves that SmNTF3 is a target gene of miRm0002.and miRm0002 regulates the expression of SmNTF3 through translational inhibition.SmNTF3 is an unknown function gene in eggplant.In order to better reveal the regulatory mechanism of miRm0002 in eggplant.SmNTF3 gene was cloned and analyzed for bioinformatics,subcellular localization,expression patterns and biological functions.Eggplant SmNTF3 gene was obtained by PCR and homologous cloning.Its open reading frame was 1119 bp in length and encoded 372 amino acids.Sequence analysis showed that SmNTF3 belonged to group C of the plant MAPK family and had high homology with NtNTF3 and SlMPK9.Subcellular localization results showed that SmNTF3 protein was localized in the nucleus and plasma membrane.Analysis of tissue expression patterns showed that SmNTF3 gene was highest expression level was in stems.Analysis of the expression pattern under salt and drought(PEG-6000)stress,SmNTF3 could be induced by these two stresses,the expression level was significantly increased,and the response under drought stress was more significant.The above results indicate that SmNTF3 may participate in the defense process of plant salt and drought stresses.In order to further study the biological function of SmNTF3,Agrobacterium-mediated inflorescence infection was used to transform Arabidopsis,and three third-generation homozygous SmNTF3 transgenic Arabidopsis lines were obtained.The effects of salt treatment on the germination rate and root length of transgenic seedlings were tested.The results showed that the germination rate of transgenic Arabidopsis thaliana seed was significantly higher than that of NaCl medium at different concentrations(50,100,150,200 mM).Wild-type seeds are lower,with significant differences at 150 mM and 200 mM concentrations.Root length observation found that the main root length of transgenic seedlings was significantly shorter than that of the wild type control at 100 and 200 mM NaCl concentration,indicating that the expression of SmNTF3 reduced Arabidopsis tolerance to salt stress.At the same time,the effects of drought on the germination rate and root length of transgenic Arabidopsis thaliana were tested using PEG-6000 simulated drought treatment.The results showed that the germination rate of transgenic seeds on 5%and 10%PEG concentration medium was higher than that of wild type,but there was no significant difference in germination rate on 15%and 20%PEG medium.The effect of PEG treatment on root length is not obvious.This result needs to be verified by further experiments.