層出鐮孢菌原生質體制備條件的研究

高美玲 劉陽 于海寧 侯紅漫 包永明

摘要[目的]研究層出鐮孢菌（Fusarium proliferatum）原生質體的最佳條件，建立高效制備層出鐮孢菌原生質體制備的方法。[方法]通過對菌絲體菌齡、酶解液組合、酶解反應溫度及酶解時間等影響因素進行優化，對層出鐮孢菌原生質體的制備條件進行研究。[結果]菌塊在CMC液體培養基中25 ℃培養分生孢子3 d后，過濾離心后轉移至YEPD液體培養基中培養12 h，該菌絲最適于層出鐮孢菌原生質體的制備；用1.2 mol/L KCl溶液配制含有5 mg/mL裂解酶、25 mg/mL崩潰酶、0.05 mg/mL溶壁酶的酶解液在30 ℃下對菌絲酶解1.5 h，此時原生質體的數量最大，為2.47×109 個/mL。原生質體再生培養時，使用40% PTC溶液恢復細胞壁，在TB3液體培養基中30 ℃過夜培養，離心后與TB3固體培養基混合后倒入平板培養，原生質體再生率可達39.75%。[結論]通過對菌絲體菌齡、酶解液濃度、溫度、酶解時間等條件的優化，可提高層出鐮孢菌原生質體的產量及再生率，為進一步研究層出鐮孢菌致病分子機制提供理論依據。

關鍵詞層出鐮孢菌；原生質體制備；原生質體再生；優化

中圖分類號S432.4文獻標識碼A文章編號0517-6611（2017）35-0149-03

Abstract[Objective] The aim was to study the optimum conditions for the preparation of protoplasts of Fusarium proliferatum， and to establish efficient methods for preparing protoplast. [Method] The preparation conditions of protoplast of Fusarium proliferatum were studied by optimizing the factors including the hypha culture time， enzyme mixtures， enzymolysis reaction temperature and enzymolysis time. [Result] The result showed that mycelia was cultured in CMC liquid medium for three days at 25 ℃， and then the conidia was transferred to YEPD liquid medium for 12 hours， which was suitable for the preparation of protoplast of Fusarium proliferatum；The enzyme mixture containing 5 mg/mL lysing enzyme， 25 mg/mL driselase and 0.05 mg/mL lywallzyme was dissolved in 1.2 mol/L KCl solution；The enzymolysis reaction temperature was 30 ℃ and enzymolysis time was 1.5 h， the yield of protoplast was the highest， and the number of protoplasts was 2.47×109 protoplasts/mL. When the protoplast was regenerated， 40% PTC solution was used to restore the cell wall， and incubated in TB3 liquid medium at 30 ℃ overnight. After centrifugation， the cells were mixed with TB3 solid medium and poured into plate culture. The protoplast regeneration rate was 39.75%. [Conclusion] The yield and regeneration rate of protoplast of Fusarium proliferatum were improved by optimizing the conditions such as hypha culture time， enzyme mixtures， enzymolysis reaction temperature and enzymolysis time， and also provide theory basis for further study on pathogenicity molecular mechanism of Fusarium proliferatum.

Key wordsFusarium proliferatum；Protoplast preparation；Protoplast regeneration；Optimization

層出鐮孢菌（Fusarium proliferatum）是一種世界范圍內導致眾多糧谷類植物病害的重要病原菌，并具有廣泛的宿主范圍，該病原菌可引起番茄葉斑病[1]、玉米穗腐病[2]、大豆根腐病[3]等。層出鐮孢菌代謝過程中產生多種有害的真菌毒素，對多種作物的食品安全性構成威脅，也是生產上較難防治的重要病害之一。由于鐮孢菌種類較多，同屬不同種鐮孢菌在生物學特性、遺傳多態性和致病機理等方面也存在較大差異。……