Department of Horticulture, Purdue University, West Lafayette, Indiana 47907.
Plant Physiol. 1989 Jan;89(1):117-25. doi: 10.1104/pp.89.1.117.
Molecular cloning of polygalacturonase (PG; EC 3.2. 1.15) from fruits of tomato (Lycopersicon esculentum Mill cv Rutgers) was accomplished by constructing a cDNA library from turning stage poly(A)(+) RNA in lambdagtll and immunoscreening with polyclonal antibodies raised against purified PG2. Both PG cDNA and antibody probes were used to quantify changes in PG gene expression in pericarp from normal, mutant, and heterozygous genotypes. Results show that PG mRNA, protein, and enzyme activity sequentially peak at the turning, ripe, and red ripe stages of Rutgers pericarp ripening, respectively. PG gene expression was attenuated greatly (0-15% of normal on a gram fresh weight basis) for PG mRNA, protein, and enzyme activity in five ripening-impaired mutants (rin, nor, Nr, Gr, and Long Keeper) tested. Maximum expression of the PG gene in heterozygotes of rin, nor, Nr, Gr, and Long Keeper (crosses with Rutgers) at the mRNA level was about 25, 13, 17, 5, and 62% of the Rutgers turning stage, at the protein level was about 166, 110, 15, 6, and 104% of the Rutgers ripe stage, and at the enzyme activity level was about 69, 37, 4, 1, and 50% of the Rutgers red ripe stage, respectively. No PG gene expression was found in preclimacteric fruits or vegetative tissues. PG mRNA was localized on both free and membrane-bound polyribosomes of ripening pericarp. In addition to transcriptional regulation, mechanisms contributing to mRNA stability, delayed protein accumulation, and posttranslational modifications may play important roles in the overall accumulation of PG activity during fruit ripening.
番茄果实多聚半乳糖醛酸酶（PG；EC 3.2.1.15）的分子克隆是通过构建一个 cDNA 文库从转阶段 poly(A)(+) RNA 在 lambdagtll 中完成的，并使用针对纯化 PG2 的多克隆抗体进行免疫筛选。PG cDNA 和抗体探针都用于定量正常、突变体和杂合基因型果皮中 PG 基因表达的变化。结果表明，PG mRNA、蛋白和酶活性分别在 Rutgers 果皮成熟的转色期、完熟期和红熟期达到峰值。在五个成熟受损突变体（rin、nor、Nr、Gr 和 Long Keeper）中，PG 基因的表达被大大减弱（以克鲜重为基础，正常的 0-15%）。rin、nor、Nr、Gr 和 Long Keeper（与 Rutgers 的杂交）杂合体 PG 基因在 mRNA 水平上的最大表达约为 Rutgers 转色期的 25%、13%、17%、5%和 62%，在蛋白水平上约为 Rutgers 完熟期的 166%、110%、15%、6%和 104%，在酶活性水平上约为 Rutgers 红熟期的 69%、37%、4%、1%和 50%。在预成熟果实或营养组织中未发现 PG 基因表达。PG mRNA 定位于成熟果皮的游离和膜结合多核糖体上。除了转录调控外，mRNA 稳定性、蛋白质积累延迟和翻译后修饰等机制可能在果实成熟过程中 PG 活性的整体积累中发挥重要作用。
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