Study On The Regulation Of MicroRNAs In Anthocyanin Biosynthesis Of Tomato

Anthocyanins are increasingly used as health-promoting components in the human diet due to their antioxidant properties.Tomato(Lycopersicum esculentum)is an economically important crop worldwide.Generally,no anthocyanins are present in cultivated tomato fruit,therefore,improving our understanding of the regulation of anthocyanin biosynthesis and enhancing anthocyanins in tomato fruits are important objective in crop improvement.Interestingly,tomato LA 1996(with anthocyanin fruit,Aft)the anthocyanin biosynthesis in fruits and seedlings is induced by light.The current research shows that except for transcription regulators and structural genes SmallRNAs are also involved in anthocyanin biosynthesis.Although the regulatory mechanism of light induced anthocyanin biosynthesis has been interpreted in detail on the level of transcription,the regulation of sRNAs involved in light dependent anthocyanin biosynthesis in tomato fruit is remaining for further study on the level of post-transcription.In this experiment,Aft tomato was used as materials,and the fruits at different development periods were treated with light or dark for 12 hours.Then total RNA was extracted from these fruit samples for small RNA sequencing and through deep sequencing analysis,miR828 and miR858 were obtained,and their target genes were also predicted and identified.The differential content of anthocyanins and transcription level of related transcription factors in different tomato varieties were detected at different development periods under light induction.The miR828-related pCAMBIA1300 recombinant plant expression vectors were constructed according to ceRNA(competing endogenous RNAs)theory and transformed into Arabidopsis and Tomatoes by Agrobacterium-mediated infiltration.The research can provide new ideas for the further study of miRNA's regulatory network for tomato anthocyanin biosynthesis.The main results are as follows:1.Three hundred and fourteen mature miRNAs were obtained through the sequencing analysis of small RNA deep sequencing data.Expression analysis screened 23 differential expressed genes and 34 differential expressed miRNAs induced by light.KEGG analysis showed the gene involved in differential expression of flavonoid synthesis pathway.2.Using RLM-5'RACE miR828 and miR858 were identified to cleave part of the predicted target genes.Different target genes had different cleavage sites,also multiple sites in one target gene were identified.3.The accumulation of anthocyanins in fruits at different stages of Aft and common edible varieties was detected under light/dark induction and we found that light can induce large amounts of anthocyanin accumulation and there aremuch accumulation in mature fruits.4.Transcription level of ten transcription factors related to anthocyanin synthesis was detected under light/dark induction conditions.The results showed that the expression of LeMYB330,LeMYB14,LeAN11,LeMYB32like was up-regulated by light-induction.5.Three plant expression vectors related to miR828 were successfully constructed:pCAMBIA1300-Si-81,pCAMBIA1300-STTM-828,and pCAMBIA1300-TAS4.Tomato fruits of Aft were used for transient gene expression by agro-infiltration system.The results showed the abundance of miR828 regulated by these transient expression genes can affect anthocyanin accumulation in peel of the injected tomatoes.6.The suitable genetic transformation screening system for Aft type tomato was optimized,we found the cotyledons with petioles can be used as explants for transformation,and the concentration of hygromycin should be 7mg/L for screening.7.The transformed Arabidopsis thaliana and Aft-type tomato were obtained by Agrobacterium-mediated method and this will provide a basis for further research of miRNA involved in anthocyanin biosynthesis.