Identification Of Mir160 Family Genes And Functional Analysis Of Csa-miR160d In Cucumber

MicroRNA（miRNA）is a group of endogenous non-coding small RNAs,which participate in various biological processes of plants by negatively regulating the expression of target genes or inhibiting their translation at the post-transcriptional level.miR160 is an evolutionally highly conserved miRNA family,which plays an important role in plant growth and development.However,little is known about miR160 in cucumber growth and fruit expansion.Based on the identification of cucumber fruit expansion gene miR160d in the early stage of the research group,bioinformatics methods were used to identify and analyze cucumber miR160 and their target gene CsARF gene family members;Csa-miR160d overexpression and short tandem simulation target（STTM）vector were constructed and introduced into Arabidopsis thaliana and cucumber to study the function of Csa-miR160d.This study provides a scientific basis for revealing the molecular mechanism of miR160 involved in the regulation of flowering and fruit expansion.The main results were as follows:1.In this study,4 Csa-MIR160 family members and 17 CsARFs were identified by genome wide searching.Gene structure analysis showed that the promoter sequences of Csa-mir160s and CsARFs contained light,plant hormone and stress response elements.CsARF5,CsARF11,CsARF13 and CsARF1 4 were predicted as the target genes of Csa-miR160s.qRT-PCR results showed that Csa-miR160d was only expressed in expanded cucumber fruits,but not in roots,leaves and unpollinated ovaries.Transcriptome data and qRT-PCR results showed that CsARF11 was expressed negatively correlated with Csa-miR160d in unpollinated ovaries and expanded fruits,which indicated that Csa-miR160d might participated in cucumber fruit expansion by nagatively regulating CsARF11.2.The over-expression and STTM vector of Csa-miR160d were constructed and transformed into Arabidopsis thaliana by dip-flowering method,and T3 transgenic plants were obtained.The results showed that,compared with wild-type Arabidopsis thaliana（WT）,plants over-expressing Csa-miR160d（Csa-miR160d_OE）showed late flowering,increased rosette（40 d）,robust plant growth,increased branch number and tall plant type（60 d）;on the contrary,inhibition of Csa-miR160d（Csa-miR160d_STTM）significantly advanced the flowering stage,significantly decreased rosette（40 d）,and resulted in slowly plant growth,decreased branch number and short plant type（60 d）.This indicated that Csa-miR160d could regulate flowering time and plant type of Arabidopsis thaliana.In addition,over-expressing Csa-miR160d promoted Arabidopsis thaliana seed germination under drought stress,while,CsamiR160d_STTM inhibited seed germination.It is speculated that Csa-miR160d might be involved in drought stress regulation.3.The over-expression and STTM vector of Csa-miR160d were transformed into cucmber ovaries（the day of anthesis without pollination）.The results showed that,compared with nontransgenic fruits（CK）,Csa-miR160d was significantly up-regulated in Csa-miR160d_OE fruits,and its target gene CsARF11 was significantly down-regulated,Csa-miR160d_OE fruits expanded rapidly in 0-5 days;on the contrary,Csa-miR160d was significantly down-regulated in Csa-miR160d_STTM fruits,and its target gene CsARF11 was significantly up-regulated,Csa-miR160d_STTM fruits were abortion from 4 d.Measurements of fruit physiological indicators showed that,compared with CK,the contents of soluble sugar and soluble protein were significantly up-regulated in Csa-miR160d_OE fruits,while significantly decreased in Csa-miR160d_STTM fruits.In addition,compared with CK,CAT and POD activities was significantly up-regulated in Csa-miR160d_OE fruits,SOD and APX activities were upregulated but not significantly,MDA was not significantly different from CK;CAT activity was significantly down-regulated in Csa-miR160d_STTM,SOD,POD and APX activities were down-regulated but not significantly,MDA was significantly higher than CK.SOD,POD,CAT,APX activities and MDA content were significantly different between Csa-miR160d_OE and Csa-miR160d_STTM fruits.These results indicated that Csa-miR160d promoted cucumber fruit expansion by inhibiting CsARF11 gene expression,increasing osmoregulatory substance content and enhancing antioxidant enzyme activity.4.Csa-miR160d_OE and Csa-miR160d_STTM cucumber transgenic plants were obtained by agrobacterium-mediated genetic transformation of cucumber cotyledon.Compared with CK,Csa-miR160d_OE promoted fruit expansion,while Csa-miR160d_STTM inhibited fruit expansion.The results of seed germination analysis showed that the seed germination rate,germination potential,main root length and lateral root number of Csa-miR160d_OE plants were significantly higher than those of CK,while the seeds of Csa-miR160d_STTM plants could not germinate.These results suggested that Csa-miR160d promoted cucumber seed germination.