海棠花青苷调控基因MsMYB44-likes的克隆与鉴定

Cloning and identification of the anthocyanin-regulated MsMYB44-likes genes in crabapple

  • 摘要: 目的 克隆和鉴定海棠花青苷生物合成相关转录因子(MYB44-likes),为揭示MsMYB44-likes基因在海棠叶片着色过程中的调控机制及海棠观赏性状改良提供参考依据。方法 以海棠品种海棠花为研究对象,对海棠组织培养幼苗进行喷施25 μmol/L脱落酸(ABA)处理,以喷施清水作为对照(CK)。PCR克隆海棠嫩叶中3个MsMYB44-likes基因(MYB44-like1MsMYB44-like2MsMYB44-like3),并对3个MsMYB44-likes基因进行生物信息学分析、编码蛋白亚细胞定位和系统发育分析。在苹果表皮中对MsMYB44-like2基因进行瞬时过表达,并通过凝胶迁移试验(EMSA)研究MsMYB44-like2蛋白与花青苷生物合成途径中结构基因海棠类黄酮糖基转移酶基因(MsUFGT)的启动子结合情况。结果 成功克隆获得3个MsMYB44-likes基因(MsMYB44-like1MsMYB44-like2MsMYB44-like3),其cDNA序列全长分别为954、960和942 bp,分别编码317、319和313个氨基酸残基;3个MsMYB44-likes蛋白结构中均以α-螺旋和无规则卷曲占比较高,且这3个蛋白与拟南芥、小麦、水稻、梨同源蛋白序列均含有典型的MYB-DNA结合结构域,MsMYB44-like1、MsMYB44-like2与梨的同源蛋白聚为一支。3个MsMYB44-likes蛋白均定位于细胞核。瞬时转化试验结果表明,注射含pCAMBIA2300-MsMYB44-like2农杆菌菌液后,苹果表皮的注射点周围呈黄绿色,说明该基因抑制苹果表皮花青苷的生物合成。与CK相比,经ABA处理后MsMYB44-like2基因的相对表达量显著降低(P<0.05)。EMSA试验结果显示,MsMYB44-like2蛋白与MsUFGT基因启动子片段的结合导致迁移率上移。结论 3个MsMYB44-likes蛋白均含有典型的MYB-DNA结合结构域,均定位于细胞核。MsMYB44-like2基因表达受ABA负调控,该蛋白通过与MsUFGT基因的启动子直接结合抑制海棠花青苷的生物合成。

     

    Abstract: Objective This study aimed to clone and identify the transcription factors (MYB44-likes) that related to the anthocyanin biosynthesis in crabapple, with the goal of providing a reference for elucidating the regulatory mechanism MsMYB44-likes genes in the leaf coloring process of crabapple and improving the ornamental traits of crabapple.Method Using the crabapple cultivar Malus spectabilis as the research object, tissue-cultured seedlings of Malus spectabilis were sprayed with 25 μmol/L abscisic acid(ABA)treatment, while the seedlings sprayed with water as the control (CK). Three MsMYB44-likes genes in the tender leaves of Malus spectabilis were cloned by PCR (MsMYB44-like1MsMYB44-like2, and MsMYB44-like3), respectively. Bioinformatics analysis, encoded protein subcellular localization and phylogenetic analysis were performed on these three MsMYB44-likes genes. Transient overexpression analysis of the MsMYB44-like2 gene was conducted in apple epidermis, and the binding of MsMYB44-like2 protein to the promoter of the structural gene UFGT in the anthocyanin biosynthesis pathway was studied using electrophoretic mobility shift assay (EMSA).Result Three MsMYB44-likes genes (MsMYB44-like1MsMYB44-like2, and MsMYB44-like3 ) were cloned, whose full-length cDNA sequences of were 954, 960 and 942 bp, and they encoded 317, 319, and 313 amino acid residues, respectively. In the three MsMYB44-likes protein structures, α-helix and random coil accounted for relatively high proportions. The protein sequences of MsMYB44-like1, MsMYB44-like2, MsMYB44-like3 and the three proteins shared typical MYB-DNA binding domains with protein sequences of Arabidopsis thaliana, wheat, rice, and pear. MsMYB44-like1 and MsMYB44-like2 clustered with the homologous proteins of pear into one clade. Three MsMYB44-likes proteins were localized in the nucleus. The transient transformation experiment showed that after injecting the Agrobacterium suspension contained pCAMBIA2300-MsMYB44-like2, the area around the injection point on the apple epidermis turned yellowish-green, indicating that the gene inhibited the anthocyanins biosynthesis of the apple epidermis. Compared with the leaves of CK, the relative expression of the MsMYB44-like2 gene in leaves after ABA treatment decreased significantly (P<0.05). The results of EMSA showed that the binding of MsMYB44-like2 protein to the promoter fragment of the MsUFGT gene led to increased mobility.Conclusion The three MsMYB44-likes proteins contain typical MYB-DNA binding domains and are localized in the nucleus. The expression of the MsMYB44-like2 gene is negatively regulated by ABA, and the protein of this gene suppresses anthocyanin biosynthesis in crabapple by directly binding to the promoter of the gene MsUFGT.

     

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