Analysis Of MiRNA And Function Of MiR398a In Chrysanthemum× Grandiflora Under Salt Stress

As a new species of ground chrysanthemum,Chrysanthemum×grandiflora has become a very important plant material in autumn landscape construction for the excavation of resistance genes.because of its strong resistance,easy management and luxuriant flowers.Plant micro RNAs are a class of 20-24 nt long non-coding s RNA that regulate important physiological processes such as growth and development,stress response and signal transduction in plants.It is the expression of plant regulation related genes that play a key role in coping with stress.However,the role of miRNA in the abiotic stress of Chrysanthemum×grandiflora is still unclear.In this study,the roots and leaves of Chrysanthemum×grandiflora treated with 200 mmol·L^-1 Na Cl（12 h）were sequenced and the differential expression miRNAs were analyzed by high-throughput sequencing.Target genes of significantly different miRNAs were predicted and annotated according to GO databases.Cloning and bioinformatics analysis of miR398a with significant difference were carried out.In order to further verify its function,an overexpression vector was constructed to genetically transform Arabidopsis thaliana,and the function of transgenic Arabidopsis thaliana was verified.The results are as follows:1.High-throughput sequencing of miRNAs was carried out in Chrysanthemum×grandiflora,and 280 miRNAs were identified,including 201 known miRNAs and 79 new miRNAs.Under salt stress there are 7 significantly differential miRNAs（3 miRNA showed up and 4 showed down）in leaf while 12 significantly differential miRNAs（6 miRNA showed up and 6 showed down）in root.Target genes of significantly different miRNAs were predicted.GO database analysis showed that a large number of miRNAs and their target genes were involved in the production of plant hormones and metabolic processes,protein binding,signal transduction pathways and so on.2.A 221 bp MIR398a was successfully cloned and named cgr-miR398a.Bioinformatics analysis showed that cgr-miR398a could form a stem-loop structure and was relatively stable,while mature miR398a had a highly conserved base sequence.3.The plant overexpression vector was successfully constructed and transformed into Arabidopsis by Agrobacterium-mediated transformation.After Kana screening and PCR detection of mature sequence,7 transgenic lines were obtained.The expression level of mature miR398a in the transgenic line was significantly higher than that in the wild type,indicating that the gene had been integrated into the genome of the plant.4.Phenotypic and physiological indicators of wild-type（WT）and over-expressing miR398a in Arabidopsis thaliana after salt,drought and stress were observed.After stress,the contents of OFR,H₂O₂ and MDA of cgr-MIR398a transgenic Arabidopsis thaliana were higher than those of wild-type,while the activities of SOD and POD were lower than those of wild-type Arabidopsis thaliana.Overexpressed,cgr-MIR398a attenuated Arabidopsis resistance to abiotic stress and played a negative regulatory role in Arabidopsis response to abiotic stress.