石榴花青苷合成相关基因PgMYB111的克隆与功能分析  

Cloning and Functional Analysis of PgMYB111 Related to Anthocyanin Synthesis in Pomegranate

作　　者：王沙 张心慧 赵玉洁 李变变 招雪晴 沈雨 董建梅 苑兆和 WANG Sha;ZHANG Xinhui;ZHAO Yujie;LI Bianbian;ZHAO Xueqing;SHEN Yu;DONG Jianmei;YUAN Zhaohe(Co-Innovation Center for Sustainable Forestry in Southern China,College of Forestry,Nanjing Forestry University,Nanjing 210037,China)

机构地区：[1]南京林业大学南方现代林业协同创新中心,南京林业大学林学院,南京210037

出　　处：《园艺学报》2022年第9期1883-1894,共12页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(31901341);江苏省自然科学基金青年基金项目(BK20180768);南京林业大学高层次人才科研启动基金项目(GXL2014070;GXL2018032);江苏省高校优势学科建设工程项目(PAPD)。

摘　　要：为研究R2R3-MYB转录因子调控石榴花青苷合成的机理,以石榴品系‘榴花红’(红花)和‘榴花白’(白花)为试材,利用同源克隆技术分离出1个调控花青苷生物合成的R2R3-MYB基因PgMYB111(Pg012177.1)。其CDS全长为1 101 bp,编码366个氨基酸。多重比对分析表明,PgMYB111蛋白的N端含有1个R2R3保守结构域,以及1个与bHLH蛋白互作的[D/E]Lx2[R/K]x3Lx6Lx3R结合位点。系统发育树分析表明,PgMYB111与苹果和扁桃具有较高的同源性,聚为一簇。亚细胞定位试验发现,PgMYB111定位于细胞核。PgMYB111在‘榴花红’中的表达量是‘榴花白’的4.3倍,且‘榴花红’花瓣花青苷含量也显著高于‘榴花白’。烟草瞬时表达结果表明转基因烟草叶片的花青苷含量和基因表达量呈先上升后下降的趋势,均在第5天达到最高,分别是未侵染叶片的3.8倍和8.9倍,pBI121空载叶片的3.17倍和5.57倍。结果表明PgMYB111参与了花青苷合成途径并促进其合成。To study the regulatory mechanism of the anthocyanin synthesis pathway in pomegranate,we isolated PgMYB111 gene from the petals of ’Liuhuahong’ and ’Liuhuabai’. Results showed that the cDNA sequence contained a 1 101 bp open reading frame(ORF)encoding a 366 amino acid polypeptide.Multiple alignment analysis of amino acid sequences showed that the N-terminus of PgMYB111 protein contained a conserved R2R3 domain and a binding site of [D/E]Lx2[R/K]x3Lx6Lx3R interacted with bHLH protein. Furthermore,the amino acid sequence had the highest similarity with MYB transcription factors in Malus × domestica and Prunus dulcis. Subcellular localization assays suggested that PgMYB111localized in the nucleus. qRT-PCR analysis indicated that the expression of Pg MYB111 gene in ’Liuhuahong’ was 4.3 times higher than that in ’Liuhuabai’. In addition,the anthocyanin content in ’Liuhuahong’ petal is significantly higher than that in ’Liuhuabai’ petal. The results of tobacco transient expression showed that the expression level of the gene increased at first and then decreased. The anthocyanin content in transgenic tobacco leaves was consistent with the gene expression,and reached the highest level on the fifth day. They were 3.8 times and 8.9 times higher than those in uninfected leaves,and 3.17 times and 5.57 times higher than those in pBI121 leaves,respectively. In summary,PgMYB111 could involve in the anthocyanin synthesis pathway and facilitate its synthesis.

关 键 词：石榴 花青苷 基因克隆 亚细胞定位 花色 

分 类 号：S665.4[农业科学—果树学]