黃毛草莓 R2R3-MYB 轉錄因子家族之全基因組鑑定及‘桃薰’果實花青素生合成

Abstract

黃毛草莓(Fragaria nilgerrensis Schltdl. ex J. Gay)為野生二倍體草莓，果實呈白色且具桃香氣，獨特果實品質及抗逆境性有益於栽培種草莓(F. × ananassa Duchesne)的育種。黃毛草莓與栽培種草莓雜交種的果實花青素含量較栽培種草莓的一般品種低，然其分子機制仍未知。R2R3-MYB為調控含花青素在內之類黃酮生合成的主要轉錄因子，本研究首先鑑定黃毛草莓全基因組之R2R3-MYB基因，比較黃毛草莓與栽培種草莓及森林草莓(F. vesca L.)類黃酮生合成相關MYB的差異，再分析種間雜交種‘桃薰’花青素生合成相關基因表現。 本研究共鑑定出91個黃毛草莓R2R3-MYB (FnMYB)，為推測其功能，首先利用MEGA 11的鄰近連接法(neighbor-joining)建立其與阿拉伯芥126個R2R3-MYB (AtMYB)之共同親緣關係樹，結果可將FnMYB分為38個亞群(F1-F38)。其中，F9、F23、F27及F11，分別對應AtMYB參與類黃酮生合成的S4、S5、S6及S7亞群。其次利用BLAST (basic local alignment search tool) 對與類黃酮生合成相關的FnMYB、栽培種草莓之FaMYB及森林草莓之FvMYB的蛋白質序列進行比對。結果顯示，FnMYB與另外兩者之序列高度相似。進一步分析DNA上啟動子序列之差異，結果顯示在調控原花青素生合成的F23/S5亞群中，FnMYB11 (evm.model.chr6.3088) 相較於FaMYB11s含有更多離層酸及茉莉酸甲酯反應相關的順式作用元件，且FnMYB11在轉錄起始點處具有長GA雙核苷酸重複序列，推測兩物種MYB11表現模式可能不盡相同。 本研究第二部分探討‘桃薰’與栽培種草莓‘臺大朱蜜’及‘TFB White’花青素生合成的差異，‘臺大朱蜜’為紅皮紅肉品種，‘TFB White’則為白皮白肉品種。試驗於溫室栽培草莓植株，採收’綠果期、白果期及成熟期3個生長階段之果實，供花青素含量測定及花青素生合成相關基因表現分析。研究發現，‘桃薰’充分成熟時果皮呈橘紅色，促進花青素生合成的MYB10表現量略低於‘臺大朱蜜’，MYB11的表現模式則與‘臺大朱蜜’相近。相較於‘TFB White’，‘桃薰’促進花青素生合成之基因表現量大部分較高。總體而言，‘桃薰’的花青素相關基因表現模式與栽培種草莓紅果品種相似。

Fragaria nilgerrensis Schltdl. ex J. Gay is a wild diploid strawberry species characterized by its white fruits with peach-like aroma. Traits such as unique fruit characteristics and stress resistance in F. nilgerrensis are valuable for the breeding of cultivated strawberry (F. × ananassa Duchesne). The interspecific hybrids between F. nilgerrensis and cultivated strawberry generally bear fruits with less anthocyanin content than the common strawberry cultivars. However, the underlying molecular mechanism remains unclear. The R2R3-MYB transcription factors (TFs) are well-known as key regulators of biosynthesis of flavonoids including anthocyanin. This study aimed to identify the R2R3-MYBs in F. nilgerrensis genome, and to compare the difference in flavonoid biosynthesis-related genes among F. nilgerrensis (FnMYBs), F. × ananassa (FaMYBs), and F. vesca L. (FvMYBs). In addition, the anthocyanin biosynthesis-related gene expression in an interspecific hybrid, ‘Tokun’, was analyzed. A total of 91 R2R3-MYB genes in F. nilgerrensis were identified in this study. To predict the functions of R2R3-FnMYB proteins, the 91 FnMYBs and 126 Arabidopsis thaliana R2R3-MYBs were integrated into a neighbor-joining tree using MEGA 11. FnMYBs were divided into 38 subgroups (F1-F38). F9, F23, F27, and F11 subgroups correspond to the S4, S5, S6, and S7 subgroups in Arabidopsis R2R3-MYB family that participate in flavonoid metabolic pathway. The protein sequence analysis through BLAST (basic local alignment search tool) showed that almost all flavonoid biosynthesis-related FnMYBs shared high levels of similarity with FaMYBs and FvMYBs. However, promoter analysis of FnMYB11 in F23/S5, the subgroup involved in proanthocyanidin biosynthesis, showed that FnMYB11 contained more abscisic acid (ABA)- and methyl jasmonate (MeJA)-responsive elements compared with FaMYB11s. Furthermore, only FnMYB11 had long GA-dinucleotide repeats spanning the transcription start site, suggesting that FnMYB11 might have different expression patterns from FaMYB11s. In the second experiment, the differences in anthocyanin biosynthesis between ‘Tokun’ and cultivated strawberry ‘NTU Jumi’ and ‘TFB White’ were investigated. ‘NTU Jumi’ is a strawberry cultivar with red skin and flesh, and ‘TFB White’ is a white strawberry cultivar. In this experiment, strawberry plants were grown in a greenhouse and harvested at green, white, and ripe fruit stages. Anthocyanin content in the fruit was measured, and the expression of genes related to anthocyanin biosynthesis was analyzed. During ripening, the fruits of ‘Tokun’ turned red-orange. The expression level of the main activator of anthocyanin biosynthesis, MYB10, was only slightly lower than that in ‘NTU Jumi’, and the expression pattern of MYB11 in ‘Tokun’ was similar to ‘NTU Jumi’. ‘Tokun’ generally had higher expression levels of genes that promote anthocyanin biosynthesis than ‘TFB White’. Overall, the expression patterns of anthocyanin biosynthesis-related genes in ‘Tokun’ fruits harvested in this study were more similar to those in the red strawberry cultivar.