摘要 | 为了解1-氨基环丙烷-1-羧酸合酶(ACS)基因在石蒜〔Lycoris radiata (L'Hér.) Herb.〕乙烯合成中的作用,对石蒜ACS基因LrACS进行了克隆,并通过系统进化树和氨基酸序列比对分析明确LrACS与其他同源蛋白的进化关系。利用亚细胞定位分析LrACS在细胞中的定位,对LrACS重组蛋白进行体外活性检测,并利用实时荧光定量逆转录PCR(qRT-PCR)分析不同生长时期LrACS的组织特异性表达。结果显示:LrACS的开放阅读框长度为1 473 bp,编码490个氨基酸。LrACS的理论相对分子质量为54 954.97,理论等电点为pI 8.21。系统进化树分析结果表明LrACS与忽地笑〔Lycoris aurea (L'Hér.) Herb.〕LaACS的亲缘关系最近,均属于Ⅰ型ACS蛋白。亚细胞定位结果显示LrACS主要定位于细胞质基质。体外活性检测结果证实LrACS重组蛋白能够催化S-腺苷甲硫氨酸(SAM)生成1-氨基环丙烷-1-羧酸(ACC)。qRT-PCR分析结果显示:LrACS在花期和叶期的石蒜各组织中均有表达,但其表达具有组织特异性,其中,花期花瓣中LrACS的相对表达量极显著(P<0.01)高于根、鳞茎、花茎,叶期根中LrACS的相对表达量极显著高于鳞茎和叶。综上所述,LrACS主要定位于细胞质基质并在石蒜不同生长时期行使催化SAM生成ACC的功能。
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Abstract | To understand the role of 1-aminocyclopropane-1-carboxylate synthase (ACS) gene in ethylene synthesis of Lycoris radiata (L'Hér.) Herb., the ACS gene of L. radiata namely LrACS was cloned, and the evolutionary relationship between LrACS and other homologous proteins was clarified through phylogenetic tree and amino acid sequence alignment analysis. The location of LrACS in the cell was determined by using subcellular localization, in vitro activity assay was performed for the LrACS recombinant protein, and the tissue-specific expression of LrACS at different growth stages was analyzed by using real-time fluorescent quantitative reverse transcription PCR (qRT-PCR). The results show that the length of open reading frame. of LrACS is 1 473 bp, encoding 490 amino acids. The theoretical relative molecular mass of LrACS is 54 954.97, and its theoretical isoelectric point is pI 8.21. The phylogenetic tree analysis result shows that LrACS has the closest genetic relationship with LaACS in Lycoris aurea (L'Hér.) Herb., and they are both of type Ⅰ ACS proteins. The subcellular localization result shows that LrACS is mainly localized in the cytoplasmic matrix. The in vitro activity assay result confirms that the LrACS recombinant protein can catalyze the conversion of S-adenosylmethionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC). The qRT-PCR analysis result shows that LrACS is expressed in all tissues of L. radiata at the flowering stage and leaf stage, but its expression exhibits tissue specificity, in which, the relative expression of LrACS in petals at the flowering stage is extremely significantly (P<0.01) higher than those in roots, bulbs, and flowering stems, and the relative expression of LrACS in roots at the leaf stage is extremely significantly higher than those in bulbs and leaves. In conclusion, LrACS is mainly localized in the cytoplasmic matrix and functions to catalyze the conversion of SAM to ACC at different growth stages in L. radiata.
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关键词 | 石蒜; 1-氨基环丙烷-1-羧酸(ACC); 1-氨基环丙烷-1-羧酸合酶(ACS); 亚细胞定位; 酶活; 功能鉴定 |
Key words | Lycoris radiata (L'Hér.) Herb.; 1-aminocyclopropane-1-carboxylic acid (ACC); 1-aminocyclopropane-1-carboxylate synthase (ACS); subcellular localization; enzyme activity; functional identification |
作者 | 樊雅婷, 李雪纯, 李晓丹, 汪仁 |
所在单位 | 江苏省中国科学院植物研究所(南京中山植物园) 江苏省植物资源研究与利用重点实验室, 江苏 南京 210014 |
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基金项目 | 江苏省植物资源研究与利用重点实验室基金项目(JSPKLB202213) |