Nieuwenhuizen Niels J, Beuning Lesley L, Sutherland Paul W, Sharma Neelam N, Cooney Janine M, Bieleski Lara R F, Schröder Roswitha, MacRae Elspeth A, Atkinson Ross G
The Horticulture and Food Research Institute of New Zealand, Mount Albert Research Centre, Private Bag 92 169, Auckland 1142, New Zealand.
The Horticulture and Food Research Institute of New Zealand, Ruakura, Private Bag 3123, Hamilton 3240, New Zealand.
Funct Plant Biol. 2007 Oct;34(10):946-961. doi: 10.1071/FP07121.
Actinidin is a cysteine protease found in Actinidia Lindl. (kiwifruit) species that affects the nutraceutical properties, processing characteristics and allergenicity of the fruit. Given the increased consumption of kiwifruit worldwide and the release of new varieties from different Actinidia species, the expression of actinidin mRNA and protein in a range of kiwifruit tissues was examined. Ten different actinidin mRNAs were identified encoding mature proteins of similar molecular weight (~24 kDa), but with predicted pIs ranging from acidic (pI 3.9) to basic (pI 9.3). In A. deliciosa 'Hayward' (green-fleshed kiwifruit) and A. chinensis 'Hort16A' and EM4 (gold-fleshed kiwifruit), actinidin mRNAs for acidic and basic proteins were expressed at comparable levels throughout ripening. Actinidin mRNA expression was highest in fruit at harvest, expression decreased as fruit ripened and was much lower in the core compared with outer pericarp tissue. Two-dimensional gel electrophoresis, combined with western analysis and liquid chromatography mass spectrometry (LC-MS) identified low levels of a novel basic actinidin protein in ripe A. deliciosa and A. chinensis fruit. Extremely high levels of an acidic actinidin protein were detected in A. deliciosa fruit and EM4, but this acidic protein appeared to be absent in 'Hort16A', the most important commercial cultivar of A. chinensis. Analyses on native gels indicated that both the basic and acidic actinidin isoforms in A. deliciosa were active cysteine proteases. Immunolocalisation showed that actinidin was present in small cells, but not large cells in the outer pericarp of mature A. deliciosa fruit at harvest. Within the small cells, actinidin was localised diffusely in the vacuole, associated with the plasma membrane, and in a layer in the plastids near starch granules. The presence of multiple forms of actinidin and varying protein levels in fruit will impact on the ability to breed new kiwifruit varieties with altered actinidin levels.
猕猴桃蛋白酶是一种在猕猴桃属植物中发现的半胱氨酸蛋白酶，它会影响果实的营养特性、加工特性和致敏性。鉴于全球范围内猕猴桃消费量的增加以及不同猕猴桃品种的推出，我们检测了一系列猕猴桃组织中猕猴桃蛋白酶mRNA和蛋白质的表达。我们鉴定出了10种不同的猕猴桃蛋白酶mRNA，它们编码分子量相似（约24 kDa）的成熟蛋白质，但预测的等电点范围从酸性（pI 3.9）到碱性（pI 9.3）。在美味猕猴桃‘海沃德’（绿肉猕猴桃）、中华猕猴桃‘Hort16A’和EM4（黄肉猕猴桃）中，酸性和碱性蛋白质的猕猴桃蛋白酶mRNA在整个成熟过程中表达水平相当。猕猴桃蛋白酶mRNA在收获时的果实中表达最高，随着果实成熟表达下降，并且与外果皮组织相比，果心处的表达要低得多。二维凝胶电泳结合蛋白质免疫印迹分析和液相色谱 - 质谱联用（LC - MS）鉴定出在成熟的美味猕猴桃和中华猕猴桃果实中存在低水平的一种新型碱性猕猴桃蛋白酶蛋白。在美味猕猴桃果实和EM4中检测到极高水平的酸性猕猴桃蛋白酶蛋白，但这种酸性蛋白在中华猕猴桃最重要的商业品种‘Hort16A’中似乎不存在。对天然凝胶的分析表明，美味猕猴桃中的碱性和酸性猕猴桃蛋白酶同工型都是活性半胱氨酸蛋白酶。免疫定位显示，在收获时成熟的美味猕猴桃果实外果皮中，猕猴桃蛋白酶存在于小细胞中，而不存在于大细胞中。在小细胞内，猕猴桃蛋白酶分散地定位在液泡中、与质膜相关以及淀粉粒附近质体的一层中。果实中多种形式的猕猴桃蛋白酶以及不同的蛋白质水平将影响培育猕猴桃蛋白酶水平改变的新猕猴桃品种的能力。
