Screening And Expression Characteristics Analysis Of Tomato MiRNA858 Target Gene MYBs

Tomato,as a daily edible fruit and vegetable,has become a model plant for research due to its small genome and short growth cycle.The Aft(Anthocyanin fruit)tomato selected for this experiment was obtained from the hybridization between L.Hilense,a wild tomato from Chile,and L.Esculentum,a cultivated tomato.In the immature stage,the fruit is accompanied by the formation of purple spots under light,which can accumulate anthocyanins.Anthocyanins play an important role in environmental adaptation,fruit development,and human health.Light is one of the most important environmental factors affecting anthocyanin synthesis.It is of theoretical and practical significance for molecular breeding to regulate metabolic pathways by changing light environmental conditions.In this study,tomato seedlings were treated with dark,blue and UV-A treatments.High-throughput sequencing technology was used to perform transcriptome sequencing,small RNA sequencing and degradation group sequencing.Differentially expressed genes were analyzed and screened,and target genes that might be involved in the regulation of light induction were mined.Transcriptome sequencing showed that the total number of differentially expressed genes in the dark and blue light comparison groups was 2010,among which the differentially expressed genes were 1452 and 558,respectively.In the dark and UV-A comparison groups,the total number of differentially expressed genes was 2008,among which the differentially expressed genes were 1443 and 565,respectively.By GO enrichment analysis,it was found that the differentially expressed genes were mainly distributed in the three processes of cell component,cell and organelle.In the molecular function classification,the differentially expressed genes were mainly distributed in binding and catalytic activity classifications.In the classification of biological processes,the differentially expressed genes are mainly distributed in the metabolic process and the cellular process.By COG functional annotation analysis,it was found that the proteins in dark and blue light and dark and UV-A differentially expressed genes had the highest frequency in general function prediction.The sequence length of small RNAs in the three samples was statistically analyzed,and it was found that the number of small RNAs with length of 24nt was the largest,followed by the number of small RNAs with length of 21nt and 22nt.We obtained 521 new miRNAs and 10 known miRNAs from the dark and blue samples,and found that 34 new miRNAs and 6 known miRNAs were differentially expressed in the two samples.We obtained 514 new miRNAs and 14 known miRNAs from the dark and UV-A samples and found that 149 new miRNAs and 14 known miRNAs were differentially expressed in the two samples.In addition,the functions of 269 target genes predicted for miRNAs were annotated.GO enrichment analysis showed that the target genes were the same in the classification of cell components,biological process and molecular functions as differentially expressed genes in transcriptome.In COG functional annotation,target genes are the same as differentially expressed genes in transcriptome,and the number of secondary metabolites in the classification of synthesis,transport and decomposition is the largest.A down-regulated gene screened by transcriptome sequencing was identified as a target gene belonging to miRNA858 by degradation group.RT-PCR confirmed that the expression level of the down-regulated gene was decreased with the development of fruit under light-induced conditions,which may be involved in regulation.GFP-SlMYB308-like vector,GFP-SlMYB86-like vector and GFP-SIMYB86-like vector were successfully constructed.After vacuum infiltration into onion epidermal cells,onion epidermal cells were observed by laser confocal microscope,indicating that the cells were located in the nucleus and cell membrane,cell membrane and nucleus,respectively.The PET-SlMYB4 vector,PET-SlMYB308-like vector,PET-SlMYB86-like vector and PET-SlMYB330 vector were successfully constructed,and finally transformed into BL21 expression state,and the obtained expression strains were named as:MYB4-BL21,MYB308-like-BL21,MYB86-like-BL21 and MYB330-BL21,respectively.By means of prokaryotic induction technique,PET-MYB308-like and PET-MYB330 were successfully induced under the induction condition of IPTG concentration of 0.1 mM at 37? for 4 h.OE-MYB4,OE-MYB308-like,OE-MYB86-like and OE-MYB330 overexpression vectors were transformed into Agrobacterium tumefacien and mixed with Agrobacterium tumefacien containing Ruby,the reporter gene regulated by the DFR promoter,to transfect tobacco leaves simultaneously.The results showed that MYB4,MYB308-like,MYB86-like and MYB330 combined with DFR promoter played a relatively small regulatory effect,which was not very significant.In the pathway of anthocyanin synthesis in plants,MYB4,MYB308-like,MYB86-like and MYB330 may be involved in anthocyanin synthesis,but their functions in the pathway of photo-induced anthocyanin synthesis in plants need to be further studied...