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[摘要]
目的 研究白桦Betula platyphylla 3-羟基-3-甲基戊二酰辅酶A 还原酶(3-hydroxy-3-methyl-glutaryl-CoA reductase,BpHMGR)全长基因结构特征及编码蛋白性质,揭示其节律表达模式以及对外源NO 信号分子的响应机制。方法 通过RACE技术克隆白桦BpHMGR 基因,应用生物信息学软件对BpHMGR 及其编码蛋白进行详细分析,利用实时荧光定量PCR 定量分析BpHMGR 基因在昼夜节律变化中的表达模式。根据BpHMGR 氨基酸序列构建系统进化树,分析昼夜节律以及外源NO对白桦叶片中BpHMGR 转录水平的影响。结果 BpHMGR 基因全长1 764 bp,包含完整开放阅读框,已上传至NCBI,并获得登录号KJ452334,其编码蛋白由587 aa 组成。白桦BpHMGR 基因与大豆(XP_003519474.1)、葡萄(XP_002275827.1)、苜蓿(XP_003617066.1)亲缘关系较近。结论 BpHMGR 随昼夜周期变化呈现出节律表达特性,在夜晚21:00 时达到峰值。初步揭示了外源NO 能诱导白桦细胞内三萜代谢途径的关键酶BpHMGR 基因上调表达,进而促进三萜产物齐墩果酸的合成。
[Key word]
[Abstract]
Objective To study the full-length gene structure characteristics and coding protein properties of 3-hydroxy-3-methylglutaryl-CoA reductase (BpHMGR) from Betula platyphylla (birch). This reveals the mechanism of rhythm expression pattern and its signaling molecule in response to exogenous NO. Methods The BpHMGR gene in birch was cloned by RACE technology. BpHMGR and its encoded protein were analyzed in detail by bioinformatics software. And the expression pattern in the circadian variation of BpHMGR gene was analyzed by real-time PCR. Results The full-length of BpHMGR gene was 1 764 bp, contained the complete ORF, encoding 587 amino acids (Genebank ID: KJ197336). According to BpHMGR amino acid sequence, phylogenetic tree was constructed. And the effect of circadian rhythm and exogenous NO on the transcription level of BpHMGR in the leaves of B. platyphylla was investigated. Conclusion BpHMGR gene, soybean (XP_003519474.1), grape (XP_002275827.1), and alfalfa (XP_003617066.1) have close genetic relationship. BpHMGR changes with the diurnal cycle showing the expression characteristics of rhythm, and reaches the peak at 9 pm. The up-regulation expression of key enzyme gene HMGR in the exogenous NO-induced birch intracellular triterpenoids metabolic pathway is initially revealed, so as to promote the synthesis of triterpene oleanolic acid product.
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[基金项目]
国家自然科学基金资助项目(J1210053,31200463);中央高校基本科研业务费专项资金资助(2572014DA04)