Mechanism Of SlMYB15 Mediated-regulation Of Cold Response In Tomato

Low temperature stress is an important limiting factor for crop growth,which can affect crop yield and quality at the same time.It is also one of the severe abiotic factors that lead to the decline of tomato(Solanum lycopersicum)yield in China.During the cold season,ensuring tomato growth and yield in solar greenhouse with limited heating system is an urgent problem for agricultural facilities in China and even the world.Based on this,the response mechanism of vegetable crops to low temperature was explored and the key factors of low temperature response were analyzed.The low temperature resistance of vegetable crops was activated by means of environmental regulation,plant growth regulators and gene editing.It has far-reaching scientific and practical significance for ensuring the annual production and commodity supply of vegetables,increasing the output of vegetables,improving the quality of vegetables and promoting the sustainable development of vegetable industry.In this paper,the main vegetable crop tomato in greenhouse culture was taken as the research object.The key factors and dynamic response changes in plants responding to low temperature in the process of low temperature treatment were identified by means of biotechnology such as biochemistry,plant physiology,molecular biology and genetics and others.Herein,the key genes of MYB transcription factor family in tomato cold resistance process induced by low temperature were screened.The interaction between MYB15 and the upstream transcription factor HY5 and genes in the CBF pathway was investigated.Key genes in the micro RNA family during low temperature treatment were screened,and the relationship between them and key genes in the MYB family was explored by transcriptional regulation analysis.Major results are shown below:1.The transcription level of 11 identified tomato MYB family genes was detected by real-time fluorescence quantitative PCR in the process of low temperature stress treatment,and the result showed that the transcription level of MYB15 was the highest up-regulated.MYB15 is localized to the nucleus by the subcellular localization fluorescence observation.MYB15 mediated low temperature tolerance of tomato was confirmed by VIGS.MYB15 overexpressed plants and CRISPR/Cas9 mutant plants were further established by using the transgenic technology,which confirmed that MYB15 plays an active regulatory role in the CBF pathway of cold tolerance of tomato.In addition,since the transcription level of MYB15 is highly correlated with CBF pathway genes and ABA pathway genes,we inferred that MYB15 is involved in hypothermia regulation in both CBF-and ABA-dependent pathways.Electrophoretic Mobility shift assay(EMSA),Chromatin Immunoprecipitation assay(Ch IP-q PCR)and Luciferase assay(LUC)confirmed that MYB15 could directly and specifically bind to promoters of CBF1,CBF2 and CBF3 genes to activate the transcription.The above results showed that low temperature induced the expression of MYB15,and MYB15 was an active regulatory factor in response to cold tolerance of tomatoes.2.The relationship among MYB15,HY5 and CBFs in the process of low temperature resistance of tomato was investigated.Low temperature can induce the increase of transcription and protein levels of HY5 and MYB15.Through the establishment of HY5 silencing plants and overexpressing plants,it was confirmed that HY5 was a positive regulatory factor in tomato,for cold tolerance.EMSA in vitro experiments,chip-q PCR and LUC in vivo experiments confirmed that HY5,as a transcription factor,can specifically recognize and bind the ACE element and G-box in the promoter sequence of genes in MYB15 and CBF pathway(such as CBF1,CBF2 and CBF3),and activate the transcription of these genes.The results of VIGS experiment show that the transcription of MYB15 is directly regulated by HY5 and other transcription factors,and that HY5 can directly or indirectly regulate the transcription of CBFs through MYB15.In the second chapter of this paper,it has been clarified that MYB15 can actively induce low temperature resistance of tomato.Synthesizing the above results,HY5-MYB15-CBFs transcriptional cascade could induce and enhance the cold tolerance of tomato.3.miR156e-3p negatively regulates the expression of MYB15 at low temperature.The expression of 13 micro RNAs in tomatoes at low temperature were detected by RT-PCR.The results showed that the expression level of miR156e-3p was significantly decreased when treated at 4 oC for 6 h,which was completely opposite to the detected expression level of MYB15 transcription factor at 4 oC.Further tobacco double luciferase assay confirmed that miR156e-3p can directly bind to the low temperature response gene MYB15 transcription factor.Overexpression of miR156e-3p inhibited the expression of MYB15 and the plants were sensitive to low temperature stress.Deletion mutant of miR156e-3p could promote the expression of MYB15 and improve the tolerance of tomatoes at low temperature.The above results showed that miR156e-3p can negatively regulate the expression of the cold stress response factor MYB15,and is a negative regulator in the tomato cold signaling pathway.