TOMATO EXO-(1-]4)-BETA-D-GALACTANASE - ISOLATION, CHANGES DURING RIPENING IN NORMAL AND MUTANT TOMATO FRUIT, AND CHARACTERIZATION OF A RELATED CDNA CLONE
TOMATO EXO-(1-]4)-BETA-D-GALACTANASE - ISOLATION, CHANGES DURING RIPENING IN NORMAL AND MUTANT TOMATO FRUIT, AND CHARACTERIZATION OF A RELATED CDNA CLONE
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DOI：
10.1104/pp.108.3.1099
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发表时间：
1995-07-01
期刊：
影响因子：
7.4
通讯作者：
SEYMOUR, GB
中科院分区：
文献类型：
--
作者：
CAREY, AT;HOLT, K;SEYMOUR, GB
使用阴离子交换、凝胶过滤和阳离子交换色谱从成熟番茄果实（Lycopersicon esculentum Mill.cv Ailsa Craig 和 cv Better Boy）中分离出外切-(1-->4)-β-D-半乳聚糖酶。最活跃级分的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳揭示了 75 kD 处的主要蛋白质条带和几个次要条带。该 75 kD 蛋白质的 30 个氨基酸 N 端序列与最近鉴定的来自柿子和苹果果实的其他 β-半乳糖苷酶/半乳聚糖酶蛋白质显示出高度的同源性（I.-K. Kang, S.-C. Suh, K.C. Gross, J.-K. Byun [1994] Plant Physiol 105: 975-979; G.S. Boss, T. Wegrzyn, E.A. MacRae, R.J, Redgwell [1994] Plant Physiol 106: 521-528) 以及由康乃馨中乙烯调节的 SR12 基因编码的预测多肽序列 (K.C. Raghothama, K.A. Lawton, P.B. Goldsbrough, W.R. Woodson [1991] Plant Mol Biol 17: 61-71）。该酶在 pH 9.8 的等电聚焦凝胶上聚焦于 P-半乳糖苷酶活性的单条带。该酶对 (1→4)-β-D-半乳聚糖底物具有特异性，最适 pH 值为 4.5。检测到的唯一反应产物是单体半乳糖，表明该酶是外切-(1-->4)-β-D-半乳聚糖酶。正常果实成熟开始时β-半乳聚糖酶活性增加，但在未成熟突变体nor 和rin 中未检测到类似的增加。使用来自康乃馨的 SR12 cDNA 克隆作为探针分离番茄同源物 (pTom beta gal 1)。该克隆在氨基酸水平上与苹果和芦笋的 β-半乳糖苷酶相关序列有 73% 的同一性，与 SR12 有 66% 的同一性。 pTom beta gal 1 是基因家族的成员。 Northern 分析表明，pTom beta gal 1 表达在正常果实中与成熟相关，在非软化突变体中表达水平较低。
An exo-(1-->4)-beta-D-galactanase was isolated from ripe tomato fruit (Lycopersicon esculentum Mill. cv Ailsa Craig and cv Better Boy) using anion-exchange, gel filtration, and cation-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most active fraction revealed a predominant protein band at 75 kD and several minor bands. A 30-amino acid N-terminal sequence from this 75-kD protein showed a high degree of homology with other recently identified beta-galactosidase/galactanase proteins from persimmon and apple fruits (I.-K. Kang, S.-C. Suh, K.C. Gross, J.-K. Byun [1994] Plant Physiol 105: 975-979; G.S. Boss, T. Wegrzyn, E.A. MacRae, R.J, Redgwell [1994] Plant Physiol 106: 521-528) and with the predicted polypeptide sequence encoded by the ethylene-regulated SR12 gene in carnation (K.C. Raghothama, K.A. Lawton, P.B. Goldsbrough, W.R. Woodson [1991] Plant Mol Biol 17: 61-71). The enzyme focused to a single band of P-galactosidase activity on an isoelectrofocusing gel at pH 9.8. The enzyme was specific for (1-->4)-beta-D-galactan substrates with a pH optimum of 4.5. The only reaction product detected was monomeric galactose, indicating that the enzyme was an exo-(1-->4)-beta-D-galactanase. beta-Galactanase activity increased at the onset of ripening in normal fruit, but no similar increase was detected in the nonripening mutants nor and rin. A tomato homolog (pTom beta gal 1) was isolated using the SR12 cDNA clone from carnation as a probe. This clone showed 73% identity at the amino acid level with beta-galactosidase-related sequences from apple and asparagus and 66% identity with SR12. pTom beta gal 1 is a member of a gene family. Northern analysis demonstrated that pTom beta gal 1 expression was ripening related in normal fruits, with lower levels apparent in the nonsoftening mutants.