Botrytis cinerea的角質酶特性及其在致病性中的作用

在以角質(cutin)爲唯一碳源的培養液中，Botrytis cinerea產生角質酶(cutinase)。酶活性在培養24天時達最大。以葡萄糖爲唯一碳源培養，不產生角質酶。當葡萄糖和角質皆存在時，角質酶的產生顯著延滯後，說明B. cinerea產生角質酶需要角質作爲誘導物，而葡萄糖對角質酶的產生有抑制的作用，將B. cinerea培養於以角質爲唯一碳素源的修定Richard培養液中20天，其濾液經硫酸銨沉澱，DEAE-52纖維素及Sephadex G-100凝膠過柱，得到純化的角質酶蛋白。某分子量爲26.2 kDa，水解反應最適pH爲8.0。B. cinerea的孢子液經紫外光誘變得一不產生酶之突變型菌株。以菌株孢子懸液接種離體草莓葉片，接種野生型菌株發病率89.3 %，接種突變型菌株發病率3.5%，在突變型菌株中加入粗角質酶液接種，發病率爲80.7%。對硫磷(parathion)可顯著抑制角質酶活性，在野生型菌株接種液中加入10μM對硫磷，發病率爲0。因此證明角質酶在B. cinerea對草莓葉片致病性中的作用。

關鍵字

角質酶；草莓

並列摘要

Botrytis cinerea isolated from the gray mold lesions of strawberry leaves was grown in shake culture in a modified Richard medium containing purified apple cutin as the sole carbon source and assayed periodically for cutinolytic activities in the culture. The maximum activity 24 days Almost enzyme detected the culture when the fungus was grown in the medium with glucose as the sole carbon source. The enzyme activities in the culture did not increase markedly until 12 day incubation when the fungus was grown with both of cutin and glucose as the carbon sources. These results showed the inducing effect of cutin and the inhibition effect of glucose on the enzyme production of cutinolytic enzyme of B. cinerea. After 20 days incubation of B. cinerea in the medium with apple cutin, the cutinase from culture filtrates was purified by ammonium sulfate precipitation, DEAE-52 cellulose in exchange chromatography, and Sephadex G-100 gel filtration. The purified enzyme had a molecular weight of 26.2 kDa as determined by SDS-poly acrylamide gel electrophoresis and had a pH optimum of 8.0 using p-nitrophenyl butyrate as substrate. A cutinase-deficient mutant was screened from the fungal conidia which were treated with ultraviolet light. The detached leaves of strawberry were inoculated with the fungal spore suspension for pathogenicity test. The disease incidence at 6 days after inoculation of wild type strain were 89.3%, but 3.5% for inoculation with the mutant. However, 80.7% of tested leaves were diseased when inoculated with mutant inoculum amended with crude extract of cutinase. The enzyme was inhibited by parathion, a inhibitor of serine esterase. No disease symptoms were observed when the wild type strain with 10uM parathion was inoculated on the leaves. These results suggested the role of cutinase in the infection of B. cinerea on the leaves of strawberry.

並列關鍵字

Botrytis cinerea ； cutinase ； strawberry

國際替代計量

Botrytis cinerea的角質酶特性及其在致病性中的作用

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