梅花花青素苷调控基因PmMYB1的分离及功能分析

Isolation and Biological Function Analysis of Anthocyanin Regulatory Gene PmMYB1 from Prunus mume

【目的】花青素苷是梅花花色形成的重要色素,R2R3-MYB是花青素苷合成调控的关键转录因子。从梅花中分离出1个R2R3-MYB调控因子PmMYB1,通过分析其序列特征并转入烟草进行功能鉴定,为探明梅花花青素苷合成调控机理及今后梅花花色分子育种奠定基础。【方法】根据梅花基因组数据设计引物,采用RT-PCR方法从梅花花瓣中分离PmMYB1基因,利用DNAMAN软件预测氨基酸序列,通过多序列比对和系统进化树构建分析其保守序列并进行功能预测,通过构建表达载体将PmMYB1基因转入烟草,分析转基因烟草的表型及花青素苷含量的变化,并利用实时荧光定量RT-PCR技术测定该基因及烟草内源花青素苷合成通路基因在转基因植株不同器官中的表达量,综合分析以上数据鉴定PmMYB1基因的功能。【结果】从梅花中分离得到全长为729 bp具有完整编码区的PmMYB1基因序列,该序列编码242个氨基酸。序列分析表明该蛋白具有保守的MYB结构域及花青素苷调控MYB蛋白特征基序,并且含有与bHLH转录因子相互作用的保守基序,可能需要bHLH蛋白协同表达共同完成花青素苷的合成调控。多序列比对和进化分析结果显示PmMYB1与其他已鉴定功能的花青素苷调控MYB蛋白有很高的同源性,其中与樱桃李的PcMYB10和欧洲甜樱桃的PaMYB10一致性分别为92%和91%。转基因烟草试验表明过表达PmMYB1基因显著提高了转基因植株花冠中花青素苷的含量(P<0.05),使其花冠颜色明显加深,实时荧光定量RT-PCR试验结果表明PmMYB1基因在转基因植株花中表达量高于叶中,过表达PmMYB1基因明显上调了烟草花中7个内源花青素苷合成通路结构基因NtCHS、NtCHI、NtF3H、NtF3′H、NtDFR、NtANS、NtUFGT及2个调控基因NtAn1a和NtAn1b的表达量。【结论】在烟草中过表达PmMYB1基因能够显著上调转基因植株花中内源花青素苷合成通路结构基因和调控基因的表达,从而促进了其花中花青素苷的积累并导致花色加深,表明该基因在花青素苷合成过程中起重要调控作用。

【Objective】 Anthocyanin is an important pigment in the flower coloration of Prunus mume. R2R3-MYB transcription factor is a key regulator of anthocyanin biosynthesis. In the present study, the PmMYB 1 gene has been isolated from P. mume.This gene was analyzed for its sequence characteristics and was transferred into tobacco to verify its biological function. This will lay a foundation for the research on the regulation mechanism of anthocyanin biosynthesis and the molecular breeding of flower color of P. mume. 【Method】 The PmMYB1 gene was isolated from the petal of P. mume using RT-PCR method. The specific primers for PCR amplification were designed according to the P. mume genome data. Multiple sequence alignment and phylogenetic tree were used to analyze the conserved sequences and predict the biological function of PmMYB1. PmMYB1 gene was transferred into tobacco by constructing expression vector. The changes of phenotype and anthocyanin content in transgenic tobacco were analyzed. In addition, qRT-PCR was used to detect the expression levels of the PmMYB 1 and the endogenous anthocyanin biosynthetic genes in different organs of transgenic tobacco. All data were analyzed to identify the function of the PmMYB1 gene.【Result】 The CDS sequence of PmMYB 1 （729 bp） were obtained by cloning from P. mume. The PmMYB1 protein encoded 242 amino acids. Sequence analysis showed that the protein had a conserved MYB domain and specific motif of anthocyanin-regulating MYB protein, and contained a conserved motif that interacts with the bHLH transcription factor, which might require the co-expression of bHLH protein in the regulation of anthocyanin synthesis. Multiple sequence alignment and phylogenetic analysis revealed that PmMYB1 was highly homologous with other identified anthocyanins-regulating MYB proteins, such as 92% and 91% identify with PcMYB10 from Prunus cerasifera and PaMYB10 from Prunus avium, respectively. Transgenic tobacco assay showed that overexpression of PmMYB1 gene significantly promoted the accumulation of anthocyanin in the corolla of transgenic plants. Compared with the control, the anthocyanin content in the flowers of transgenic plants was higher（ P 〈 0.05）, which caused to the deepening of flower color. The qRT-PCR assay showed that the expression level of PmMYB1 gene was higher in the flowers of transgenic plants than that of leaves. The expression level of seven structural genes （ NtCHS, NtCHI, NtF 3H, NtF3′ H, NtDFR, NtANS, and NtUFGT ）and two regulatory genes（ NtAn1a and NtAn1b ） of anthocyanin biosynthesis in the flowers of transgenic lines were obviously up-regulated compared with the control.【Conclusion】Overexpression of PmMYB1 in tobacco significantly promoted anthocyanin accumulation in the flowers and caused to the deepening of flower color of transgenic plants through activating endogenous structural and regulatory genes of anthocyanin biosynthesis to up-regulate their expression in the flowers of transgenic lines.These findings suggest that PmMYB1 plays an important role in the regulation of anthocyanin synthesis.