Gapper Nigel E, Coupe Simon A, McKenzie Marian J, Scott Richard W, Christey Mary C, Lill Ross E, McManus Michael T, Jameson Paula E
New Zealand Institute for Crop & Food Research Limited, Food Industry Science Centre, Private Bag 11600, Palmerston North, New Zealand.
Institute of Molecular BioSciences, Massey University, Private Bag 11222, Palmerston North, New Zealand.
Funct Plant Biol. 2005 Oct;32(10):891-901. doi: 10.1071/FP05076.
To gain an in-depth understanding of the role of ethylene in post harvest senescence, we used broccoli (Brassica oleracea var. italica) as our model species. The senescence-associated asparagine synthetase (AS) promoter from asparagus was used to drive the expression of an antisense 1-aminocyclopropane-1-carboxylate oxidase (ACO) cDNA from broccoli, BoACO2, to reduce ethylene production following harvest. Physiological analyses revealed that transgenic broccoli lines harbouring the antisense BoACO2 gene construct (designated as AS-asACO) displayed delayed senescence in both detached leaves and detached heads as measured by hue angle. Harvested floret tissue from these plants also showed a delayed loss of chlorophyll, lower protease activity and higher total protein content, and changes in transcript levels of senescence marker genes when compared with wild type and transgenic lines transformed with an empty T-DNA. Genes that were down-regulated included those coding for cysteine protease (BoCP5), metallothionein-like protein (BoMT1), hexokinase (BoHK1), invertase (BoINV1) and sucrose transporters (BoSUC1 and BoSUC2). Northern analysis for BoACO1 and BoACO2, ACO assays and western analysis, revealed reduced ACO transcript, enzyme activity and protein accumulation, as well as reduced ethylene production in the transgenic AS-asACO lines when compared with controls, confirming that a key enzyme regulating ethylene biosynthesis was reduced in these plants. This, together with the changes observed in gene expression, confirm a significant role for ethylene in regulating the events leading to senescence in broccoli following harvest.
为了深入了解乙烯在采后衰老中的作用，我们选用西兰花（Brassica oleracea var. italica）作为模式物种。来自芦笋的衰老相关天冬酰胺合成酶（AS）启动子用于驱动来自西兰花的反义1-氨基环丙烷-1-羧酸氧化酶（ACO）cDNA（BoACO2）的表达，以减少采后的乙烯生成。生理分析表明，携带反义BoACO2基因构建体（命名为AS-asACO）的转基因西兰花品系，通过色相角测量，在离体叶片和离体花球中均表现出衰老延迟。与野生型和用空T-DNA转化的转基因品系相比，这些植株收获的小花组织还表现出叶绿素损失延迟、蛋白酶活性较低、总蛋白含量较高，以及衰老标记基因转录水平的变化。下调的基因包括编码半胱氨酸蛋白酶（BoCP5）、类金属硫蛋白（BoMT1）、己糖激酶（BoHK1）、转化酶（BoINV1）和蔗糖转运蛋白（BoSUC1和BoSUC2）的基因。对BoACO1和BoACO2的Northern分析、ACO测定和蛋白质免疫印迹分析表明，与对照相比，转基因AS-asACO品系中ACO转录本、酶活性和蛋白质积累减少，乙烯生成也减少，证实这些植株中调节乙烯生物合成的关键酶减少。这与观察到的基因表达变化一起，证实了乙烯在调节西兰花采后导致衰老的过程中起重要作用。
