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MicroRNA828(miRNA828)是一种新近发现的生物学功能还未全面研究的miRNA。为从不同角度阐明miRNA828的生物学功能,从拟南芥中克隆到At-pri-miR828基因并构建了该基因过量表达的植物表达载体pC2300-pOT2-At-pri-miR828,通过农杆菌介导的叶盘法将pC2300-pOT2-At-pri-miR828导入异源植物番茄品种‘Ailsa Craig’中。PCR鉴定结果显示,外源基因At-pri-miR828已成功整合到转基因番茄基因组中,共获得9个转基因株系,67株转基因植株。定量PCR检测结果显示,与野生型番茄植株相比,转基因植株中miR828的表达量显著增加,而生物信息学所预测的miR828靶基因Sly-myb-like1的表达水平则相应降低。花青素含量测定结果显示,miR828过量表达的转基因番茄植株花青素含量明显低于野生型植株,表明miR828参与了番茄花青素的生物合成调控。
MicroRNA828 (miRNA828) is a recently discovered miRNA that has not yet been fully investigated for its biological function. To elucidate the biological functions of miRNA828 from different angles, At-pri-miR828 gene was cloned from Arabidopsis thaliana and the over-expression plant expression vector pC2300-pOT2-At-pri-miR828 was constructed and transformed into Agrobacterium PC2300-pOT2-At-pri-miR828 was introduced into the heterotic plant tomato cultivar ’Ailsa Craig’ by the leaf disc method. PCR analysis showed that the exogenous gene At-pri-miR828 has been successfully integrated into the genome of transgenic tomato, and a total of 9 transgenic lines and 67 transgenic plants were obtained. Quantitative PCR results showed that the expression level of miR828 in transgenic plants was significantly increased compared with wild type tomato plants, while the expression level of miR828 target gene Sly-myb-like1 predicted by bioinformatics was correspondingly decreased. The results of anthocyanin assay showed that anthocyanin content of transgenic tomato plants overexpressing miR828 was significantly lower than that of wild type plants, indicating that miR828 is involved in the regulation of anthocyanin biosynthesis in tomato.