CAJ | 학술논문

【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。【目的】花青素苷是月季花瓣呈紅色或粉色的重要因素。R2R3-MYB 蛋白是調控植物花青素苷閤成的關鍵轉錄因子。從月季花瓣中剋隆花青素苷調控相關的 R2R3-MYB 蛋白同源基因，併分析這些基因與月季花瓣顏色和花青素苷閤成的關繫，為花色基因工程改良奠定基礎。【方法】根據植物花青素苷調控相關 R2R3-MYB 蛋白的保守序列設計簡併引物，結閤3'-RACE 和 Y-RACE 方法從月季花瓣中擴增同源基因的全長編碼序列。用植物第四（Sg4）和第六（Sg6）亞傢族的 R2R3-MYB 蛋白、擬南芥次生代謝調控相關的 R2R3-MYB 序列和剋隆基因的編碼蛋白進行多序列比較和進化分析。通過比較不同顏色的月季花瓣中花青素苷含量和 R2R3-MYB 蛋白基因的錶達水平，分析月季花瓣中 R2R3-MYB 蛋白基因與花青素苷調控的關繫。【結果】從月季‘紅勝利’的紅色花瓣中剋隆瞭2箇 R2R3-MYB 蛋白基因（RhMYBs4-1和RhMYBs6-1，GenBank登錄號分彆為KJ664810和KJ664811）。序列分析錶明，RhMYBs4-1和RhMYBs6-1蛋白均含有保守的 R2R3-MYB 結構域，分彆與植物 Sg4和 Sg6 R2R3-MYB 蛋白同源。RhMYBs4-1具有 Sg4 MYB 蛋白典型的 C1、C2抑製子和鋅指結構。RhMYBs6-1具有 Sg6 MYB 蛋白特有的（A/S/G）NDV 和 KPRPR（T/S）基序。錶達分析顯示 RhMYBs4-1和 RhMYBs6-1均在月季‘紅勝利’的花瓣中高水平錶達，在葉片和花藥中錶達水平很低。比較7種不同顏色月季花瓣中的花青素苷含量和 RhMYBs4-1和 RhMYBs6-1的錶達水平，髮現紅色花瓣中花青素苷含量遠高于其他材料，相應地，RhMYBs4-1和 RhMYBs6-1均在紅色花瓣中具有最高的錶達水平。在粉紅色月季花瓣中，花青素苷含量不到紅色花瓣的10%，RhMYBs4-1的錶達水平極低，而 RhMYBs6-1的錶達水平與紅色花瓣相噹。【結論】月季 RhMYBs4-1和 RhMYBs6-1分彆編碼 Sg4和 Sg6亞傢族的 R2R3-MYB 蛋白，均在紅色月季花瓣中高水平錶達，可能是月季花青素苷閤成和花瓣顏色的重要調控基因。
【목적】화청소감시월계화판정홍색혹분색적중요인소。R2R3-MYB 단백시조공식물화청소감합성적관건전록인자。종월계화판중극륭화청소감조공상관적 R2R3-MYB 단백동원기인，병분석저사기인여월계화판안색화화청소감합성적관계，위화색기인공정개량전정기출。【방법】근거식물화청소감조공상관 R2R3-MYB 단백적보수서렬설계간병인물，결합3'-RACE 화 Y-RACE 방법종월계화판중확증동원기인적전장편마서렬。용식물제사（Sg4）화제륙（Sg6）아가족적 R2R3-MYB 단백、의남개차생대사조공상관적 R2R3-MYB 서렬화극륭기인적편마단백진행다서렬비교화진화분석。통과비교불동안색적월계화판중화청소감함량화 R2R3-MYB 단백기인적표체수평，분석월계화판중 R2R3-MYB 단백기인여화청소감조공적관계。【결과】종월계‘홍성리’적홍색화판중극륭료2개 R2R3-MYB 단백기인（RhMYBs4-1화RhMYBs6-1，GenBank등록호분별위KJ664810화KJ664811）。서렬분석표명，RhMYBs4-1화RhMYBs6-1단백균함유보수적 R2R3-MYB 결구역，분별여식물 Sg4화 Sg6 R2R3-MYB 단백동원。RhMYBs4-1구유 Sg4 MYB 단백전형적 C1、C2억제자화자지결구。RhMYBs6-1구유 Sg6 MYB 단백특유적（A/S/G）NDV 화 KPRPR（T/S）기서。표체분석현시 RhMYBs4-1화 RhMYBs6-1균재월계‘홍성리’적화판중고수평표체，재협편화화약중표체수평흔저。비교7충불동안색월계화판중적화청소감함량화 RhMYBs4-1화 RhMYBs6-1적표체수평，발현홍색화판중화청소감함량원고우기타재료，상응지，RhMYBs4-1화 RhMYBs6-1균재홍색화판중구유최고적표체수평。재분홍색월계화판중，화청소감함량불도홍색화판적10%，RhMYBs4-1적표체수평겁저，이 RhMYBs6-1적표체수평여홍색화판상당。【결론】월계 RhMYBs4-1화 RhMYBs6-1분별편마 Sg4화 Sg6아가족적 R2R3-MYB 단백，균재홍색월계화판중고수평표체，가능시월계화청소감합성화화판안색적중요조공기인。
[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.