协同抑制番茄ACO1对果实成熟及病程相关蛋白基因表达的影响

The Influence of Co-suppressing Tomato 1-Aminocyclopropane-1 -Carboxylic Acid Oxidase I on the Expression of Fruit Ripening-Related and Pathogenesis-Related Protein Genes

目的探讨协同抑制番茄ACO1基因对果实成熟和病程相关蛋白基因表达、内源乙烯生物合成及果实耐贮性的影响。方法采用PCR或RT-PCR方法克隆了番茄ACC氧化酶1、ACC氧化酶3、EBF1、PR1、PR5以及NP24基因片段,并以此制备探针,以协同抑制ACO1的转基因番茄和野生型番茄为研究对象,进行Northern杂交,同时测定了伤害叶片和果实的乙烯释放量,并进行了果实贮藏试验等。结果Northern杂交结果表明,番茄ACO1基因表达被抑制后,与果实成熟相关基因LeACO3和LeEBF1,以及病程相关蛋白基因LePR1、LePR5和LeNP24的表达量急剧降低。乙烯释放量测定试验和果实贮藏试验结果表明,协同抑制LeACO1番茄完整和受伤叶片以及完整果实内源乙烯释放量相对于野生型番茄大大减少,成熟果实贮藏时间延长。结论协同抑制番茄ACO1基因表达的同时,与果实成熟相关基因和病程相关蛋白基因的表达也不同程度地受到抑制,而且其内源乙烯生物合成减少,果实耐贮性增强。

[Objective] The purpose of this study is to explore the influence of co-suppressing tomato ACC oxidase I on the expression of fruit ripening-related and pathogenesis-related protein genes and on the biosynthesis of endogenous ethylene and storage ability of fruits. [ Method] Specific fragments of several fruit ripening-related and pathogenesis-related protein genes from tomato （Lycopersicon esculentum） were cloned, such as the l_aminocyclopropane-l-carboxylic acid oxidase Ⅰ gene （LeAC01）, 1-aminocyclopropane-l-carboxylic acid oxidase Ⅲ gene （LeAC03）, EIN3-binding F-box 1 gene （LeEBF1）, pathogenesis-related protein 1 gene （LePR1）, pathogenesis-related protein 5 gene （LePR5） and pathogenesis-related protein osmotin precursor gene （LeNP24） by PCR or RT-PCR. Then these specific DNA fragments were used as probes to hybridize with the total RNAs extracted from the wild type tomato （AC^＋＋） and the LeACO1 co-suppression tomatoes （VII87 and T4B）, respectively. At the same time, ethylene production measurement and storage experiment of tomato fruits were carried out. [Result] The hybridization results indicated that the expression of fruit ripening-related genes, such as LeAC03 and LeEBF1, and pathogenesis-related protein genes, such as LePR1, LePR5 and LeNP24, were reduced sharply. The ethylene production in the fruits and wounded leaves decreased.The storage time of ripening fruits was prolonged, when the expression of LeACO1 gene in the transgenic tomato was suppressed. [Conclusion] In the co-suppression tomatoes, the expression of fruit ripening-related and pathogenesis-related protein genes were restrained at the different degrees, the biosynthesis of endogenous ethylene decreased and the storage ability of tomato fruits increased.