Identification And Regulation Functions Of MiRNAs Involed In Anthocyanin Synthesis In Mulberry

Mulberry anthocyanins are natural water-soluble pigments,and many structural genes and regulatory genes are involved in their biosynthesis pathways.As a kind of important regulatory factor,plant miRNA plays important regulatory roles in anthocyanin biosynthesis.In this study,mulberry fruits at different growth stages were used as materials to analyze the expression profiles of miRNAs.Using small RNA digitization analysis techniques based on Illumina high-throughput sequencing,the mi RNAs involved in mulberry anthocyanin biosynthesis were identified,and the target genes of the differentially expressed miRNAs were predicted by psRNATarget software.The regulations of the differentially expressed mi RNAs and their target genes in the process of anthocyanin biosynthesis were studied using transgenic and qRT-PCR techniques.The results obtained in this study will lay foundation for revealing the molecular mechanism of mulberry anthocyanin synthesis,and also provide candidate genes for genetic improvement of mulberry,and promote the research on the functional genomics of mulberry.The main findings are as follows:1.Detection of anthocyanins of mulberry fruits at different growth stagesThe content of total anthocyanins of mulberry fruits at different growth stages was determined by a pH differential method.Moreover,the qualitative and quantitative analyses of anthocyanins were performed with HPLC method.The results showed that the anthocyanins of mulberry fruits were mainly composed of cyanidin-3-O-glucoside and cyanidin-3-O-rutoside glycosides which content was increased when the color of mulberry fruits changed from green to red and then to purple.2.Construction and sequencing of mulberry small RNA high-throughput sequencing libraryThe small RNA sequencing libraries of mulberry fruits at different growth stages were constructed and sequenced.A total of 139 conserved miRNAs belonging to 51 families were identified,and 42 new miRNAs were predicted.Among the miRNAs identified,there were 76 mi RNAs were differentially expressed in the mulberry fruits at different growth stages.There were 124 genes were predicted to be the targets of the differentially expressed miRNAs,and most of these targets were transcription factors involved in plant secondary metabolic biosynthesis.3.Cloning of Mul-MIR159 a and its target gene Mul-MYB33 and their regulation functions in anthocyanin synthesisThe Mul-MIR159 a gene and its target gene Mul-MYB33 were cloned by PCR,and their plant expression vectors were constructed,respectively.Moreover,the transgenic Arabidopsis plants of Mul-MIR159 a and Mul-MYB33 were obtained successfully.The results of qRT-PCR indicated that mul-miR159 a can target the Ath-MYB33 which is the homologous gene of Mul-MYB33 in the transgenic Arabidopsis plants.Phenotypic analysis of transgenic Arabidopsis plants showed that overexpression of Mul-MIR159 a gene in Arabidopsis reduced the anthocyanin accumulation in the transgenic plants,whereas overexpression of Mul-MYB33 gene in Arabidopsis increased the accumulation of anthocyanins in the transgenic plants,indicating that the Mul-MIR159 a had a negative regulation on the biosynthesis of anthocyanins.4.Cloning and expression activity analysis of the promoters of Mul-MIR159 a and Mul-MYB33The promoters(pMul MIR159a)of Mul-MIR159 a and the Mul-MYB33(pMulMYB33)were cloned using PCR technique,respectively,The plant expression vectors of the two promters were constructed.Using PlantCARE online software,the promoter core elements TATA and CAAT-Box were found in both the two promoters.In addition,light,hormones,and low temperature response elements and MYB binding sites were also found in the two promoter sequences.The inducing expression activities of the two promoters were analyzed by transient infection of tobacco using GUS histochemical staining methods,and the results showed that the activities of the two promoters can be induced by light and GA.In addition,it was showd that the Mul-MIR159 a and Mul-MYB33 can be induced by light and GA.5.Regulation founctions of mul-mi R477—Mul-ABCB19-AS—Mul-ABCB19 cascade in anthocyanin synthesisThe Mul-MIR477,Mul-ABCB19-AS,and Mul-ABCB19 genes were cloned by PCR,and their plant expression vectors were constructed,respectively.Then the mul-miR477—Mul-ABCB19-AS—Mul-ABCB19 cascade was established in Arabidopsis successfully.It was demonstrated that Mul-ABCB19-AS can be cleaved by mul-miR477,and Mul-ABCB19-AS can silence the expression of the Mul-ABCB19 gene.It was showed that overexpression of Mul-ABCB19 gene in Arabidopsis increased the accumulation of anthocyanin in transgenic plants,and the mul-miR477 had a positive regulation on the biosynthesis of anthocyanin mediated by the mul-miR477—Mul-ABCB19-AS—Mul-ABCB19 cascade.