滇牡丹PdMYB57基因克隆及功能验证

Cloning and functional verification of PdMYB57 from Paeonia delavayi

【目的】滇牡丹作为牡丹育种材料,具有丰富的花色资源,研究其MYB转录因子对花色的调控作用可为牡丹花色分子育种工作提供帮助。【方法】以黄色及红色滇牡丹花部组织为材料进行徒手切片,并以其转录组数据为基础进行序列比对,得到1个MYB转录因子编码基因PdMYB57。经基因克隆、系统进化树构建、定量逆转录聚合酶链式反应(qPCR)、瞬时表达及高效液相色谱(HPLC)等验证该基因功能。【结果】PdMYB57基因有1个798 bp的完整开放阅读框,编码265个氨基酸,为亲水性不稳定蛋白;与拟南芥SG6亚族及卵叶牡丹PqMYB113、牡丹PsMYB57/PsMYB58及葡萄VvMYBA1/VvMYBA2等MYB转录因子聚为一簇,具有R2R3保守结构域[R/K]Px[P/A/R]xx[F/Y]基序;其在红色花滇牡丹萼片及黄色花滇牡丹叶片中高表达;其瞬时表达后能使烟草叶片产生紫色,HPLC表明其瞬时表达的烟草叶片中含有矢车菊素3-O-芸香糖苷(Cy3R)。【结论】PdMYB57基因编码1个R2R3-MYB转录因子,其表达具有组织特异性,并能促进植物花青素合成。

[Objective]As a peony breeding material,Paeonia delavayi has abundant flower color resources.Studying the regulatory effects of its MYB transcription factors on flower color will benefit molecular breeding of peony flower color.[Methods]Using the flowers of yellow and red P.delavayi as materials for hand sectioning.Based on the transcriptomic data of P.delavayi,a MYB transcription factor coding gene PdMYB57 was obtained through sequence alignment.Function of this gene was verified through gene cloning,phylogenetic tree construction,qRT-PCR,transient expression,and HPLC.[Results]PdMYB57 gene has an ORF of 798 bp,encoding an unstable hydrophilic protein of 265 amino acids.PdMYB57 was clustered with the Arabidopsis SG6 subfamily,as well as MYB transcription factors in P.qiui PqMYB113,P.suffruticosa PsMYB57/PsMYB58,and Vitis vinifera VvMYBA1/VvMY BA2.PdMYB57 had a R2R3 conserved domain[R/K]Px[P/A/R]xx[F/Y]motif.PdMYB57 gene was highly expressed in sepals of red P.delavayi and leaves of yellow P.delavayi.HPLC analysis showed that tobacco leaves transiently expressed PdMYB57 gene contained cyanidin-3-O-rutinoside(Cy3R).[Conclusion]PdMYB57 gene encodes an R2R3-MYB transcription factor.PdMYB57 gene expression is tissue-specific and promotes anthocyanin synthesis in plants.