石榴种皮木质素合成相关转录因子基因PgMYB的克隆与表达

Clone and Expression of a Lignin Biosynthesis-related Transcription Factor Gene PgMYB in Pomegranate Seed Coat

为初步探讨石榴(Punica granatum L.)籽粒硬度产生机理及转录因子基因PgMYB在石榴种皮木质素生物合成途径中的作用,测定了不同石榴品种籽粒硬度及种皮总木质素含量并分析两者关系,利用RT-PCR结合RACE技术,克隆了‘红玉石籽’石榴的1个MYB转录因子基因(PgMYB),通过实时荧光定量PCR技术分析了PgMYB的相对表达量。结果表明:(1)石榴籽粒硬度与种皮总木质素含量呈显著正相关关系,相关系数为0.906。(2)PgMYB基因cDNA全长1 088bp,开放阅读框921bp,编码蛋白由306个氨基酸组成,N端具有2个MYB DNA结合结构域,是植物中一个典型的R2R3-MYB转录因子;同源分析显示,该基因编码的氨基酸序列与银合欢的MYB1和拟南芥的MYB4一致性分别高达89%和84%。(3)在不同籽粒硬度石榴品种中PgMYB的表达与籽粒硬度和种皮总木质素含量呈负相关关系。(4)在石榴各个发育时期中,PgMYB表达与种皮总木质素含量同样呈负相关关系。推测该基因可能抑制石榴种皮总木质素的生物合成。

To study the mechanism of pomegranate seed hardness, and the function of transcription factor gene PgMYB in lignin biosynthesis pathway of pomegranate seed coat,we measured the seed hardness and total lignin content in different pomegranate cultivars. A novel MYB transcription factor gene PgMYB was isolated from cultivar ＇Hongyushizi＇ by reverse transcription-polymerase chain reaction （RT-PCR） and rapid amplification of cDNA ends （RACE）. The expression levels of PgMYB were analyzed by real time- PCR. The results showed that： （1）The hardness of pomegranate seed and the total lignin content in seed coat were positively correlated with a correlation coefficient of 0. 906. （2）The full-length cDNA of PgMYB was 1 088 bp with an open reading frame of 921 bp encoding a protein of 306 amino acid residues. It＇s a typical R2R3-MYB transcription factor gene in plant with two MYB DNA binding domains at its N-termi- nus. Blast X analysis showed that PgMYB had high identity with Leucaena leucocephala MYB1 and Arabi- dopsis thaliana MYB4 as 89% and 84% ,respectively. （3）Real time-PCR analysis indicated that the relative expression of PgMYB was negatively correlated with the hardness of pomegranate seed and total lignin content in seed coat. （4）At each stage of ＇ Hongyushizi＇ fruit development,the relative expression of Pg MYB was always negatively correlated with the total lignin content in seed coat. It was speculated that Pg MYB might repress the biosynthesis of lignin in pomegranate seed coat. The results expected to lay a foun dation for study the mechanism of pomegranate seed hardness.