| Tomato（Solanum lycopersicum）is one of the Solanaceae family,which is an economically valuable crop.Late blight infestation can seriously affect tomato quality and cause devastating damage.Therefore,it is important to improve the tomato self-defense ability and cultivate disease-resistant varieties from the level of molecular biology.Micro RNA（miRNA）have been abundantly demonstrated to be critical factors in the stress response of plants.In addition,previous studies have shown that short peptides encoded by miRNA primary can promote the expression level of corresponding miRNA,thereby exerting its regulatory function.Therefore,this study used genetic engineering technology,miRNA and gene function identification and other methods,combined with bioinformatics analysis,to explain the regulatory mechanism of mi R399,ubiquitin conjugating enzyme E2 24（UBC24）and its primary encoded peptide（mi PEP）on tomato in late blight resistance.The main findings are as follows:（1）To explore the regulatory mechanism of tomato mi R399 in late blight resistance.Bioinformatics methods were used to determine the target genes of mi R399 and analyze the expression characteristics.An engineered strain of mi R399 overexpression and Short tandem target mimic silencing（STTM）vector was created and treated with transient infestation of late blight fungus,respectively.The results showed that transient overexpression of mi R399decreased the expression of its target gene UBC24,increased the expression of pathogenesis related protein（PR）gene,and affected the expression level of Jasmonic acid（JA）signaling pathway gene,the opposite was true for transient silencing of mi R399.Preliminary insights into the role of mi R399 as a positive regulator.（2）To explore the regulatory mechanism of target gene UBC24 in late blight resistance.The silencing vector of UBC24 was constructed by using virus induced gene silencing（VIGS）technology to create an Agrobacterium tumefaciens engineering strain.The results showed that transient silencing of UBC24 reduced the content of reactive oxygen species（H2O2and O2-）in tomato leaves,which could improve tomato resistance to late blight.And it could also regulate tomato defense responses by affecting the expression of disease resistance-related genes PRs and transcript levels of genes regulating the JA signaling pathway affect resistance to late blight in tomato.（3）To mining and preliminary identification of tomato mi R399 primary coding peptide sequence,and research the disease resistance.To analyze the coding peptide sequence of mi R399 primary by bioinformatics methods,and two overexpression vectors（p BI121-mi PEP399-1 and p BI121-mi PEP399-2）of tomato mi PEP399 were constructed by plant high-efficiency overexpression technology.An engineered strain of overexpression vector was created and treated with transient infestation of late blight fungus.The results showed that transient overexpression of tomato mi PEP399-2 increased the expression level of mi R399,decreased the expression level of UBC24 and affected the expression of disease resistance-related genes PRs,suggesting that mi PEP399-2 has biological activity.Transient overexpression of tomato mi PEP399-1 didn’t promote the expression of mi R399,suggesting that mi PEP399-1 has no biological activity.It was preliminarily demonstrated that mi PEP399-2 could promote the expression of mi R399 and improve the resistance of tomato to late blight.In conclusion,this study verified the regulatory mechanism of mi R399 and its target gene UBC24 in the process of tomato resistance to pathogenic bacteria infection,mined mi R399 primary coding peptide sequences,and preliminarily identified their biological activity.It provides a theoretical basis for molecular disease resistance breeding of tomato,and also provides a reference for the related research on peptides encoded by plant miRNA primary. |
