Identification And Functional Analysis Of Mirna Involved In Cucumber(cucumis Sativus L.) Fruit Expansion

MicroRNA?miRNA?is an important class of regulatory molecules in plants.miRNA is involved in a series of biological processes,such as growth and development,hormone signal transduction and response to abiotic and biotic stresses by regulating gene expression at post-transcriptional level either by cleavage or translational inhibition of targeted mRNA.However,the regulatory mechanisms of miRNA in cucumber fruit expansion are unclear.In this study,a small RNA?sRNA?and degradation group library created from cucumber fruit were constructed,and the miRNA and target genes involved to cucumber fruit expansion were obtained.Bioinformatics analysis of CsamiR160 d and CsaMIR319 b,which were significantly expressed in expanding cucumber fruit,and their target genes were carried out.The functions of CsamiR160 d and CsaMIR319 b in cucumber plant growth and fruit expansion were revealed by over expression of miRNA.This study provided a theoretical basis and important genetic resources for understanding the miRNA-mediated regulatory mechanisms controlling fruit expansion in cucumber.The main findings are as follows:1.Three sRNA libraries and degradation group libraries related to cucumber fruit expansion at different development stages were constructed.A total of 2522 miRNAs belonging to 66 families were identified in the sRNA library,of those,58 known miRNAs and 47 novel miRNAs exhibited highly differentially expression in the fruiton five days post anthesis with pollination?EXP₅d?.Expression patterns of 11 highly differentially expressed miRNAs were validated by quantitative real-time PCR?qRT-PCR?,the expression patterns were similar to sRNA sequencing data,which confirmed the reliability of sRNA sequencing data.The degradation group sequencing identified miRNA target genes.GO analysis found that the target genes of the differentially expressed miRNAs were enriched in biological processes,cell components and molecular functions.KEGG analysis showed that these target genes were mostly found to be associated with environmental adaptation,signal transduction and translation.After comprehensive analysis,CsamiR160 d and CsaMIR319 b were selected for further study.2.Bioinformatics analysis of CsamiR160 d and its target genes CsARF17 and CsARF18 were carried out in this study.The results showed that the upstream promoter element of CsamiR160 d is related to fruit growth and maturity,and tCsARF17 and CsARF18 gene family are functionally conserved.It is speculated that CsamiR160 d regulates the expansion of cucumber fruits involved in the auxin signaling pathway by targeting ARF.Nine over-expressed CsamiR160 d cucumber plants were obtained and the transformation frequency was 28.13%.Compared to non-transgenic cucumber plants,over-expression of CsamiR160 d showed stronger growth vigour,such as higher plant height,thicker stem and much more leaves.This indicated that over-expression of CsamiR160 d promotes the growth of cucumber plants.3.Bioinformatics analysis of CsaMIR319 b and its target genes CsDME were carried out in this study.The results showed that the upstream promoter element of CsaMIR319 b is related to fruit growth and maturity,as well as,they are involved in plant stress response.The function of the CsDME family is also conservative.It is speculated that CsaMIR319 b may affect the DNA methylation of cucumber by regulating its target gene CsDME,and then affect the fruit expansion of cucumber.Four over-expressed CsaMIR319 b transgenic plants were obtained,and the transformation frequency was 40.00%.Compared to non-transgenic cucumber plants,over-expression of CsaMIR319 b suppressed the plant growth,fruit development and transgenic seed germination.This indicated that over-expression of CsaMIR319 b inhibited plant growth and fruit expansion.