月季花青素苷相关R2R3-MYB蛋白基因的克隆和表达分析

doi:10.3864/j.issn.0578-1752.2015.07.14

摘要/Abstract

摘要： 【目的】花青素苷是月季花瓣呈红色或粉色的重要因素。R₂R₃-MYB蛋白是调控植物花青素苷合成的关键转录因子。从月季花瓣中克隆花青素苷调控相关的R₂R₃-MYB蛋白同源基因，并分析这些基因与月季花瓣颜色和花青素苷合成的关系，为花色基因工程改良奠定基础。【方法】根据植物花青素苷调控相关R₂R₃-MYB蛋白的保守序列设计简并引物，结合3'-RACE和Y-RACE方法从月季花瓣中扩增同源基因的全长编码序列。用植物第四（Sg4）和第六（Sg6）亚家族的R₂R₃-MYB蛋白、拟南芥次生代谢调控相关的R₂R₃-MYB序列和克隆基因的编码蛋白进行多序列比较和进化分析。通过比较不同颜色的月季花瓣中花青素苷含量和R₂R₃-MYB蛋白基因的表达水平，分析月季花瓣中R₂R₃-MYB蛋白基因与花青素苷调控的关系。【结果】从月季‘红胜利’的红色花瓣中克隆了2个R₂R₃-MYB蛋白基因（RhMYBs4-1和RhMYBs6-1，GenBank登录号分别为KJ664810和KJ664811）。序列分析表明，RhMYBs4-1和RhMYBs6-1蛋白均含有保守的R₂R₃-MYB结构域，分别与植物Sg4和Sg6 R₂R₃-MYB蛋白同源。RhMYBs4-1具有Sg4 MYB蛋白典型的C1、C2抑制子和锌指结构。RhMYBs6-1具有Sg6 MYB蛋白特有的（A/S/G）NDV和KPRPR（T/S）基序。表达分析显示RhMYBs4-1和RhMYBs6-1均在月季‘红胜利’的花瓣中高水平表达，在叶片和花药中表达水平很低。比较7种不同颜色月季花瓣中的花青素苷含量和RhMYBs4-1和RhMYBs6-1的表达水平，发现红色花瓣中花青素苷含量远高于其他材料，相应地，RhMYBs4-1和RhMYBs6-1均在红色花瓣中具有最高的表达水平。在粉红色月季花瓣中，花青素苷含量不到红色花瓣的10%，RhMYBs4-1的表达水平极低，而RhMYBs6-1的表达水平与红色花瓣相当。【结论】月季RhMYBs4-1和RhMYBs6-1分别编码Sg4和Sg6亚家族的R₂R₃-MYB蛋白，均在红色月季花瓣中高水平表达，可能是月季花青素苷合成和花瓣颜色的重要调控基因。

关键词: 月季, 花瓣颜色, 花青素苷, R₂R₃-MYB蛋白, 基因表达

Abstract: 【Objective】Anthocyanin is a major factor conferring pink or red to rose flower. R₂R₃-MYB proteins are key transcription factors controlling anthocyanin biosynthesis. This work aimed to clone homologous genes of anthocyanin-related R₂R₃-MYB protein from rose flowers, and further to analyze their relatedness to anthocyanin biosynthesis and coloration in rose petals, which may lay a foundation for gene engineering improvement of flower.【Method】With a degenerate primer designed according to the conserved sequence of anthocyanin-related R₂R₃-MYB proteins from various plants, complete coding sequences of corresponding homologous genes were amplified and cloned by using 3′-RACE and Y-RACE methods. Multiple sequence alignment and phylogenetic analysis were performed using typical sequences from the 4th and 6th subfamily of plant R₂R₃-MYB proteins (Sg4 and Sg6) and Arabidopsis R₂R₃-MYB proteins related to secondary metabolism with deduced proteins of the cloned genes. To elucidate the relatedness of the cloned genes to anthocyanin biosynthesis and coloration in rose petals, anthocyanin contents and expression levels of the two cloned R₂R₃-MYB protein genes were determined in rose petals of different colors. 【Result】Two R₂R₃-MYB protein genes (RhMYBs4-1 and RhMYBs6-1, GenBank accession Nos, KJ664810 and KJ664811, respectively) were cloned from red petals of rose ‘Red Success’. Sequence analyses indicated that RhMYBs4-1 and RhMYBs6-1, both conserved in the R₂R₃-MYB domain, were homologous to Sg4 and Sg6 R₂R₃-MYB proteins, respectively. RhMYBs4-1 contained the C1, C2 repressors and zinc finger domains typical of Sg4 R₂R₃-MYB proteins, whereas RhMYBs6-1 had the signature motifs ((A/S/G)NDV and KPRPR(T/S)) of Sg6 R₂R₃-MYB subfamily. Expression analyses showed that both RhMYBs4-1 and RhMYBs6-1 were expressed at a high level in the red petals of rose ‘Red Success’, but at low levels in leaves and stamens. Among rose petals of different colors, red petals had the highest anthocyanin contents, and the highest expression levels of both RhMYBs4-1 and RhMYBs6-1 genes. In pink rose petals, the anthocyanin content was less than 10% of that in red petals and the RhMYBs4-1 expression level was very low, whereas the expression level of RhMYBs6-1 in pink petals was comparable to that in red petals. 【Conclusion】Results of this study indicated that the RhMYBs4-1 and RhMYBs6-1 genes encoded Sg4 and Sg6 R₂R₃-MYB proteins, respectively. Both RhMYBs4-1 and RhMYBs6-1 were highly expressed in red petals and might be important regulators of anthocyanin biosynthesis and coloration in rose petals.

Key words: Rosa hybrida, petal color, anthocyanin, R₂R₃-MYB protein, gene expression

赵佳，刘荣，杨帆，李鑫，刘厚生，严倩，肖月华. 月季花青素苷相关R₂R₃-MYB蛋白基因的克隆和表达分析[J]. 中国农业科学, 2015, 48(7): 1392-1404.

ZHAO Jia, LIU Rong, YANG Fan, LI Xin, LIU Hou-sheng, YAN Qian, XIAO Yue-hua. Cloning and Expression Analyses of R₂R₃-MYB Genes Related to Anthocyanin Biosynthesis in Rose[J]. Scientia Agricultura Sinica, 2015, 48(7): 1392-1404.

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月季花青素苷相关R₂R₃-MYB蛋白基因的克隆和表达分析

Cloning and Expression Analyses of R₂R₃-MYB Genes Related to Anthocyanin Biosynthesis in Rose

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