Expression of Recombinant Aequorin as an Intracellular Calcium Reporter in the Phytopathogenic Fungus Phyllosticta ampelicida

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• 作者：Shaw ; Brian D. ; Kozlova ; Olga ; Read ; Nick D. ; Turgeon ; B. Gillian ; Hoch ; H. C.
• 刊名：Fungal Genetics and Biology
• 出版年：2001
• 出版时间：December, 2001
• 年：2001
• 卷：34
• 期：3
• 页码：207-215
• 全文大小：149 K

文摘

Conidia of Phyllosticta ampelicida germinate only after they have made contact with a substratum. Previous work has shown that external free calcium must be available to the spore for germination to be initiated. Transgenic strains of P. ampelicida expressing apo-aequorin, a calcium-sensitive luminescent protein, were developed to monitor cytoplasmic free Ca²⁺ ([Ca²⁺]b>cb>). Transformants were verified by PCR and Southern hybridization. Apo-aequorin production was quantified for each of 21 transformants. The transformant that emitted the most light per unit of protein was found to contain 0.59 mg apo-aequorin/g total protein. To ascertain the feasibility of aequorin-based [Ca²⁺]b>cb> quantification, [Ca²⁺]b>cb> changes were measured in mycelia during various physiologically perturbing treatments: exposure to high concentrations of external Ca²⁺, hypoosmotic shock, and mechanical perturbation. This is the first report of a plant pathogenic fungus for which aequorin-based Ca²⁺ measurement protocols have been developed.