Characterization of CRISPR Mutants Targeting Genes Modulating Pectin Degradation in Ripening Tomato
Characterization of CRISPR Mutants Targeting Genes Modulating Pectin Degradation in Ripening Tomato
复制标题
DOI：
10.1104/pp.18.01187
复制
发表时间：
2019-02-01
期刊：
影响因子：
7.4
通讯作者：
Seymour, Graham B.
中科院分区：
文献类型：
--
作者：
Wang, Duoduo;Samsulrizal, Nurul H.;Seymour, Graham B.
番茄（Solanum lycopersicum）是一种全球重要作物，经济价值达数百亿美元，是人类饮食中必需维生素、矿物质和植物化学物质的重要供应者。保质期是与角质层特性的改变和水果细胞壁的重塑相关的关键质量特性。过去 20 年对转基因番茄植物的研究表明，一系列果胶降解酶参与细胞壁重塑。这些研究通常只涉及单个基因的沉默，并且事实证明很难比较不同实验系统中沉默这些基因的效果。在这里，我们报告了编码果胶降解酶果胶裂解酶（PL）、多聚半乳糖醛酸酶2a（PG2a）和β-半乳聚糖酶（TBG4）的成熟相关基因中基于CRISPR的突变体的产生。对一系列 PL、PG2a 和 TBG4 CRISPR 品系的果实的理化特性进行比较表明，尽管 PG2a 和 TBG4 的突变影响果实的颜色和重量，但只有 PL 的突变才会导致果实更坚硬。分别使用单克隆抗体探针 LM19 去酯化的同型半乳糖醛酸、INRA-RU1 到鼠李半乳糖醛酸 I、LM5 到 β-1,4-半乳聚糖和 LM6 到阿拉伯聚糖表位，研究了 CRISPR 突变水果果皮细胞中果胶的定位、分布和溶解度。数据表明 PL、PG2a 和 TBG4 作用于不同的细胞壁结构域，并且纤维素微纤维相关果胶的重要性反映在其在不同突变系中出现的增加。
Tomato (Solanum lycopersicum) is a globally important crop with an economic value in the tens of billions of dollars, and a significant supplier of essential vitamins, minerals, and phytochemicals in the human diet. Shelf life is a key quality trait related to alterations in cuticle properties and remodeling of the fruit cell walls. Studies with transgenic tomato plants undertaken over the last 20 years have indicated that a range of pectin-degrading enzymes are involved in cell wall remodeling. These studies usually involved silencing of only a single gene and it has proved difficult to compare the effects of silencing these genes across the different experimental systems. Here we report the generation of CRISPR-based mutants in the ripening-related genes encoding the pectin-degrading enzymes pectate lyase (PL), polygalacturonase 2a (PG2a), and beta-galactanase (TBG4). Comparison of the physiochemical properties of the fruits from a range of PL, PG2a, and TBG4 CRISPR lines demonstrated that only mutations in PL resulted in firmer fruits, although mutations in PG2a and TBG4 influenced fruit color and weight. Pectin localization, distribution, and solubility in the pericarp cells of the CRISPR mutant fruits were investigated using the monoclonal antibody probes LM19 to deesterified homogalacturonan, INRA-RU1 to rhamnogalacturonan I, LM5 to beta-1,4-galactan, and LM6 to arabinan epitopes, respectively. The data indicate that PL, PG2a, and TBG4 act on separate cell wall domains and the importance of cellulose microfibril-associated pectin is reflected in its increased occurrence in the different mutant lines.