Molecular Mechanism Of Mir396b-5p Regulating Cold Tolerance In Cucumber/pumpkin Grafted Seedlings

Grafting is one of the key agronomic measures to guarantee the production of cucumbers(Cucumis sativus L.)in cold period.miRNA may play an important role in the response of grafted seedlings to stress,but a large number of studies have focused on the isolation and identification of differentially expressed miRNA in grafted complexes,and few studies on the molecular regulatory mechanisms mediated by them.In this study,the cucumber variety Jinchun 4"(Cucumis sativus L.)was used as the scion and the "black seed pumpkin"(Cucurbita ficifolia)as the rootstock.The high-throughput sequencing technology was used to isolate and identify the differentially expressed miRNA in the leaves of the grafted seedlings.and the qRT-PCR technology was used to analyzed the response characteristics of these differentially expressed miRNAs to low temperature stress,and the most abundantly expressed miR396b-5p was identified and responded to low temperature.Through genetic transformation of Arabidopsis thaliana,the role of miR396b-5p and its target genes in low temperature stress were analyzed.To explore the molecular mechanism of miR396b-5p regulating the cold tolerance of cucumber/pumpkin grafted seedlings,which is of great significance to enrich the theory of horticultural plant grafting and lay a theoretical foundation for the resistance regulation of grafting complexes.The main findings are as follows:1.Grafting can significantly improve the cold tolerance of grafted cucumber seedlings.After 7 days of low temperature treatment,the decrease in fresh weight and dry weight of grafted seedlings was significantly lower than that of non-grafted seedlings and selfgrafted seedlings,while the cold tolerance index,leaf soluble sugar content and Pro content were significantly higher than the latter,but the MDA content was significantly lower than non-grafted seedlings and self-grafted seedlings.2.The differentially expressed miRNAs in the leaves of grafted cucumber/pumpkin seedlings play an important physiological role.High-throughput sequencing analysis found that there were 60,66,and 61 differentially expressed miRNAs in the three combinations of non-grafted and self-grafted(NG-vs-SG),non-grafted and hetero-grafted(NG-vs-CG)and self-grafted and hetero-grafted SG-vs-CG.Among the differentially expressed miRNAs,miR166 and miR408,miR395,miR399 and miR156 were confirmed to be involved in the response of grafted seedlings to low temperature stress.Their target gene functions are involved in a variety of biological processes including growth and development,photosynthesis,wound healing,nutrient transport(such as sulfur and phosphorus,etc.),hormone signal transduction etc.3.MIR396 gene(pre-miR396b-5p)Arabidopsis Overexpressed mainly enhance the cold resistance through the ICE-CBF-COR gene regulation network system.The expression level of mature miR396b-5p was significantly increased,the corresponding target gene GRF family members were down-regulated and FLU expression was up-regulated in pre-miR396b-5p overexpressed Arabidopsis.The expression level of ATCBF1,ATCOR15A,RD29A three cold-resistant genes increased significantly in pre-miR396b-5p overexpressed Arabidopsis,which mainly acted on the ICE-CBF-COR gene regulatory network system to enhance the cold resistance of transgenic Arabidopsis.