Construction And Transformation Of MiRNAs Of Anthocyanin Biosynthesis In Tomato

MicroRNAs (miRNAs) are endogenous21-24nucleotide single—stranded RNAs and play key roles in various biological processes, such as, gene expression, development, cell cycles and environmental stress in plants. Anthocyanin is a kind of water-soluble and naturally edible pigment. Anthocyanins have beneficial health effects but are not highly present in some important crop plants, such as tomato fruits. Thus, tomatoes highly enriched with anthocyanins will enhance the health-promoting quality. miR828and miR858are newly identified and their functions are still unknown. The potential target genes for miR828and miR858are PAP1/MYB75and MYB12respectively predicted by miRBase and TargetScan. Over-expression of MYB75and MYB12can activate the whole biosynthetic pathway of anthocyanin. In this study, in order to explore the function of miR828in anthocyanin biosynthesis, the following experiments were done.1. The pri-miR828gene was cloned from Arabidopsis thaliana with PCR approach and transferred into the corresponding site of the pCAMBIA2300in the sense orientation, and generated the vector of gain-of-function mutants of pri-miR828—pC2300-pOT2-pri-miR828. Antother vector of loss-of-function mutants of the miR858—pC2300-pOT2-STTM858was generated by short tandem target mimicry (STTM) technologies.2. Then, the two vectors were introduced into tomato by Agrobacterium. The positive transgenic plants were identified by PCR technique. The results showed that the targeted fragment was amplified from all the tested transgenic plants except for one among the eight At-pri-miR828transgenic lines.3. The At-pri-miR828gene relative expression level detection was carried out though semi-quantitative RT-PCR analysis. It turned out that exogenous gene showed differential expression patterns in each tomato plant. This test laid the foundation for the follow-up further study the function of miR828in tomato.