Functional Analysis Of MiR166 And MiR319 In Response To Salt Stress In Cucumber

Cucumber(Cucumis sativus L.)is an important crop in protected horticulture.However,soil salinity hinders its production in greenhouses.MicroRNA(miRNA)can act on target genes to regulate plant resistance against abiotic stresses.In our previous studies,we have found some differentially expressed microRNAs under salt stress in cucumber through analysis of high-throughput sequencing technology data.Further,we investigate the functions of these key miRNAs in response to salt stress.In order to provide some bases for improvement in salt tolerance of cucumber and to clarify the mechanism of miRNA during salt stress,we have screened one or more miRNAs that could play a critical role in salt stress tolerance mainly by transgenic technology.The major results were as followed:1.By analyzing the high-throughput sequencing data and qRT-PCR results,we screened out miR166 and miR319 as two major miRNAs that are responsible for salt stress tolerance in cucumber.Ten differentially expressed miRNAs were obtained from the previous sequencing data of our group and the target genes related to salt stress were selected as the predictive target genes of these 10 miRNAs.Furthermore,the relationship between miRNAs and predicted target genes were tested by qRT-PCR.The results showed that miR166 negatively regulates its predicted target genes in the leaves and root tips.However,miR319 negatively regulates its predicted target genes in the root tips of cucumber.2.At first,homozygous transgenic lines of Arabidopsis thaliana with miR166 and miR319 were obtained.The DNA of 'Jinchun No.2' cucumber was used as a template to clone precursor genes related to miR166 and miR319.Then,we constructed an overexpression vector by inserting pre-miR166 and pre-miR319 and transferred them to Arabidopsis thaliana respectively.Finally,homozygous transgenic Arabidopsis thaliana of T3 generation was obtained through resistance screening.3.The identification results from the phenotype,physiology and gene expression level showed that the salt tolerance of transgenic Arabidopsis thaliana could be improved by miR166,and could be alleviated by miR319 in the seedling stage of Arabidopsis thaliana.When transgenic Arabidopsis thaliana was treated with different concentrations of salt,the growth of miR166 and miR319 plants was improved in terms of germination rate,cotyledon greening rate and root length as compared to the wild-type Arabidopsis thaliana.Moreover,when these plants were treated with 300mM NaCl solution,the survival rate of transgenic Arabidopsis thaliana was higher than that of wt.However,when they were exposed to 200mM salt stress,miR166 and miR319 transgenic Arabidopsis showed a lower concentration of Na+and a higher concentration of K+as compare to their wt.By detecting the expression level of key salt-tolerant genes in Arabidopsis thaliana under salt stress,we have found that the expression levels of HKT1,NHX1,SOS3,and AVP1 were increased in roots.However,an increment in NHX1 and SOS1 was observed in leaves of miR166 and miR319 transgenic Arabidopsis thaliana as compare to the wt The results showed that the salt tolerance of transgenic Arabidopsis thaliana with miR166 and miR319 was better than that of wild-type.4.The Csa7G452940 gene was proved to be a target gene of miR166 by tobacco transient transformation system.The four predicted target genes of miR166 were verified by the transient transformation system in tobacco.The GFP fluorescence was only disappeared when the bacterial solution of miR166 and Csa7G452940 target sites were injected into tobacco leaves,which indicates that Csa7G452940 gene is the target gene of miR166.