Identification And Characterization Of MicroRNAs Involved In Blue And UV-A Light-induced Anthocyanin Biosynthesis In Turnip

Anthocyanin is a kind of flavonoid compounds that are major plant phenylpropanoid metabolites found throughout the plant kingdom.Light is a key environmental factor to affect anthocyanin biosynthesis by inducing the structural and regulatory gene expression in the plant anthocyanin biosynthesis pathway.MicroRNAs are a class of non-coding small RNAs,which exert crucial roles in the regulation of plant growth and development,as well as in the stress resistance.Recent studies have revealed that some microRNAs are responsive to light signals while some others could regulate anthocyanin biosynthesis and accumulation.However,the roles of miRNAs involved in light-induced anthocyanin biosynthesis pathway remain unknown.Herein,we took the seedlings of Brassica rapa turnip'Tsuda',a light-dependent anthocyanin biosynthesis plant,as the experimental material.After being treated by dark,blue and UV-A light,total RNA was extracted and small RNA libraries were constructed.Then Illumina/Solexa deep sequencing was employed to identify the microRNAs response to blue and UV-A light and involved in anthocyanin biosynthesis.Afterwards,the targets of these microRNAs were predicted,their expression levels were detected and the cleavage sites on their target genes were identified.Next,the precursors of several microRNAs and the complete sequence of several target genes were cloned,inserted into pBI1Z1 plasmid to form a recombinant vector and subjected to Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral-dip method.Our work aimed at providing clues for further studying on the roles of microRNAs in light-induced anthocyanin biosynthesis pathway.The main results are listed as follows:1:Deep small RNA-seq was performed and 58 conserved miRNAs,235 less-conserved miRNAs and 412 novel miRNA candidates were obtained.2:Stem-loop qRT-PCR and northern blot analysis of several conserved miRNAs and novel miRNA eandidates revealed that their expression levels were basically consistent with the standard expression level of deep sequencing.3:The 43 conserved miRNAs had been predicted to target 273 genes representing a wide range of enzymes,transcription factors and other proteins with vital functions.4:QRT-PCR analysis of 7 light-induced differentially expressed miRNAs and their putative target genes suggested that only some of them negatively correlated upon light irradiation,meanwhile,these putative target genes encoded proteins involved in many important life processes.5:QRT-PCR analysis of miR157?miR828 and their putative target genes which may play a part in anthocyanin accunulation demonstrated that miR157 could down-regulate SPL9 and SPL15 upon blue arid UV-A irradiation,while there was an inverse correlation between miR828 and three MYB genes,PAPl,PAPZ and MYB82.6:RLM-5' RACE identified that the accurate cleavage sites of miR157?miR390 and miR828 were at the 10th or 11th base from the 5' ends of miRNA sequences in their binding region,and mostly at the 10th base.7:The precursors of miR157?miR390 and miR828 were cloned and could form stem-loop structures after being analyzed by RNA Folding Form.8:The complete sequence of turnip coding gene BrSPL9,non-coding genes BrTAS3 and BrTAS4 which may have a function in anthocyanin biosynthesis pathway in turnip,were cloned by RT-PCR.9:Recombinant plasmids pBI121-PreMiR157?PBI121-BrSPL9?PBI121-BrTAS3and pBI121-BrTAS4 were successfully constructed and applied to Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral-dip method.The T1 seeds of the pBI121-PreMiR157 transformant have been harvested and used for kanamycin resistance screening,which lay a foundation for further study.