Identification And Biological Function Analysis Of Genes Involed In Anthocyanin Synthesis In Mulberry

Anthocyanins are a kind of natural water-soluble pigments with important physiological activities.The synthesis pathways of anthocyanins in Arabidopsis,grape,petunia and other plants have been reported.However,the biosynthesis of anthocyanins is a complex biological process,involving the participation of many structure genes and regulatory genes,and the specific molecular regulatory mechanisms in the process of biosynthesis are unknown yet.In this study,the expression profiles of mRNAs and lncRNAs in mulberry at different developmental stages were analyzed,and the differential expression of mRNAs and lnRNAs involved in the synthesis of mulberry anthocyanins were identified.Moreover,the regulatory networks of the gene identified were analyzed.The regulation mechanism and function of the genes involved in anthocyanin synthesis were studied by transgenic technology,and the role and regulation mechanism of these genes in anthocyanin biosynthesis pathway were revealed.The information provided here will be great significance to reveal the molecular control mechanism of anthocyanin synthesis pathway comprehensively,and it will provide candidate genes for mulberry genetic improvement and lay a foundation for functional genomics research of mulberry.The main results of this study are as follows:(1)Construction and sequencing analysis of the high throughput transcriptome sequencing library of mulberry fruits.The RNA libraries of green mulberry fruits(MG),red mulberry fruits(MR)and purple mulberry fruits(MP)were successfully constructed and sequenced with Illumina Solexa sequencing.A total of 153620 transcripts,106233 mRNAs and 24226 lncRNAs were identified.Among the genes identified,there were 10230 differentially expressed mRNAs and 4219 differentially expressed lncRNAs in mulberry fruits at different developmental stages.In addition,there were 211 differentially expressed mRNAs and 243 differentially expressed lncRNAs,which were related to anthocyanin biosynthesis were identified.(2)Analysis of the function and expression regulation of Mul-UC3 GT gene in anthocyanin biosynthesis in mulberry fruits.Based on the information of mulberry fruit transcriptomes,the Mul-UC3 GT gene was cloned using PCR technology.The plant expression vector of Mul-UC3 GT was constructed and the transgenic Arabidopsis thaliana plants were obtained.It was showed that overexpression of Mul-UC3 GT gene in Arabidopsisincreased the accumulation of anthocyanin in transgenic plants.In addition,the DFR and LDOX genes were up-regulated while the UFGT and UGT75 genes were down-regulated in the transgenic plants.It has been showed that Mul-UC3 GT was one target of mul-miR472.To explore the regulation functions of mul-miR472,the artificial miRNA Mul-aMIR472 was constructed by overlap PCR technology and the plant expression vector of its was constructed and then was transformed into Arabidopsis thaliana to obtain the transgenic Arabidopsis thaliana plants.Moreover,the hybrids between Mul-UC3 GT and Mul-aMIR472 transgenic Arabidopsis were produced to analyze the cleavage of Mul-UC3 GT transcript by mul-miR472.The fluorescence quantitative PCR analysis showed that mul-miR472 could target Mul-UC3 GT gene in the hybrid seedlings,which down regulate the expression of Mul-UC3 GT gene expression.Further analysis showed that the accumulation of anthocyanin in the hybrid seedlings was decreased.Therefore,mul-miR472 could regulate the expression of Mul-UC3 GT gene in post transcriptional level and had a negative regulation on the biosynthesis of anthocyanins in mulberry.(3)Regulation of Mul-CHS-AS and Mul-CHS on anthocyanin biosynthesis.High throughput sequencing analysis showed that the lncRNA,Mul-CHS-AS,was an antisense of Mul-CHS gene.In order to study the role of Mul-CHS-AS and Mul-CHS in the synthesis of anthocyanin,the Mul-CHS-AS and Mul-CHS gene was cloned,respectively.In addition,the plant expression vectors of the two genes were constructed and their transgenic plants were obtained.It was showed that the accumulation of anthocyanins in Mul-CHS transgenic Arabidopsis thaliana was significantly higher than that in the wild type Arabidopsis thaliana.In addition,the C4 H and 4CL genes were down-regulated while the CHI and F3 H genes were up-regulated in the transgenic plants.Moreover,the Mul-CHS transgenic Arabidopsis thaliana and Mul-CHS-AS transgenic Arabidopsis were hybridized to obtain the hybrid seedlings.Quantitative PCR analysis showed that the expression of Mul-CHS gene in the hybrid seedlings was significantly lower than that in the Mul-CHS transgenic Arabidopsis thaliana.Phenotypic analysis and anthocyanin content determination showed that the accumulation of anthocyanin in the hybrid seedlings was also reduced,indicating that the Mul-CHS-AS gene can regulate the expression of the Mul-CHS gene and has a negative regulation on anthocyanin biosynthesis.