重寄生真菌盾壳霉胞外蛋白酶产生条件及酶活影响因子

doi:10.16409/j.cnki.2095-039x.2016.03.017

中国生物防治学报 ›› 2016, Vol. 32 ›› Issue (3): 406-413.DOI: 10.16409/j.cnki.2095-039x.2016.03.017

重寄生真菌盾壳霉胞外蛋白酶产生条件及酶活影响因子

谢晓莉^1,2, 杨龙¹, 吴明德¹, 张静¹, 李国庆¹

1. 作物病害监测与安全控制湖北省重点实验室/华中农业大学植物科学技术学院, 武汉 430070;
2. 西安市雁塔区食品药品监督管理局, 西安 710061

收稿日期:2015-10-26 出版日期:2016-06-08 发布日期:2016-06-07
通讯作者: 杨龙
作者简介:谢晓莉(1985-),女,硕士,E-mail:xiexiaoli77@163.com
基金资助:
国家自然科学基金(31471813,31000877);高校博士点基金新教师基金(20100146120036)

Culture Condition and Characterization of Factors Affecting Activity of the Extracellular Proteases Produced by Mycoparasite Coniothyrium minitans

XIE Xiaoli^1,2, YANG Long¹, WU Mingde¹, ZHANG Jing¹, LI Guoqing¹

1. Hubei Key Laboratory of Plant Pathology/College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China;
2. Xi'an Yanta District Food and Drug Administration, Xi'an 710061, China

Received:2015-10-26 Online:2016-06-08 Published:2016-06-07

摘要/Abstract

摘要： 重寄生真菌盾壳霉Coniothyrium minitans是核盘菌Sclerotinia sclerotiorum的重要生防菌。为了探讨盾壳霉胞外蛋白酶在寄生核盘菌过程中的作用,采用明胶平板法对盾壳霉寄生核盘菌菌核产生的蛋白酶活性进行了检测,并进一步采用福林酚法定量测定蛋白酶活性,研究盾壳霉产生胞外蛋白酶的培养条件及影响蛋白酶活性的因子。试验结果表明,在被盾壳霉寄生的核盘菌菌核中检测到蛋白酶活性,表明蛋白酶可能参与盾壳霉重寄生作用。发现核盘菌菌核浸出液培养基适合盾壳霉产生胞外蛋白酶,摇培(20℃、200r/min)5d时蛋白酶活性最高,达到0.22U/mL。盾壳霉胞外蛋白酶酶促反应的最适温度为60℃,最适pH7.0。当温度不高于40℃时,蛋白酶酶活较稳定。5mmol/L的金属离子Mg²⁺、Zn²⁺、Ca²⁺、Cu²⁺、Mn²⁺、Li⁺和K⁺等对蛋白酶酶活没有显著影响(P>0.05),而Fe²⁺(5mmol/L)显著(P<0.05)提高了蛋白酶活性。盾壳霉蛋白酶对苯甲基磺酰氟(PMSF)敏感,说明盾壳霉产生的胞外蛋白酶可能主要是丝氨酸蛋白酶。这些结果为盾壳霉胞外蛋白酶的分离纯化和功能研究奠定了基础。

关键词: 盾壳霉, 核盘菌, 重寄生, 蛋白酶

Abstract: The mycoparasite Coniothyrium minitans is an important biocontrol agent of Sclerotinia sclerotiorum. In order to elucidate the role of the extracellular proteases (EPs) produced by C. minitans in mycoparasitism on S. sclerotiorum, a study was carried out to detect the activity of the EPs produced by C. minitans using the methods of gelatin-displying and the Folin phenol reagent quantification, and to clarify the role of the EPs in mycoparasitism of C. minitans. Results showed that the protease activity was detected in C. minitans-infected sclerotia of S. sclerotiorum, implying that the EPs may get involved in mycoparasitism of C. minitans. Results also showed that S. sclerotiorum sclerotia extracts significantly enhanced production of the EPs by C. minitans, compared to the media without S. sclerotiorum sclerotia extracts. In the time-course experiment, production of the EPs by C. minitans in the shake cultures (20 ℃, 200 r/min) was peaked at 5 days post incubation and the protease activity reached up to 0.22 U/mL. The optimum enzymatic reaction conditions were: temperature at 60 ℃, pH at 7.0. The activity of the EPs was stable bellow 40 ℃. Presence of Mg²⁺, Zn²⁺, Ca²⁺, Cu²⁺, Mn², Li⁺, K⁺ (5 mmol/L each) had no significant (P>0.05) effect on the activity of the EPs. However, the presence of Fe²⁺ at 5 mmol/L significantly (P<0.05) enhanced the EP activity. The EPs were highly sensitive to phenylmethyl sulfony fluride (PMSF), implying that C. minitans EPs might belong to the serine protease family. These results will be useful for the future study to purify the C. minitans EPs and to clarify the role of the EPs in biocontrol of C. minitans against S. sclerotiorum.

Key words: Coniothyrium minitans, Sclerotinia sclerotiorum, mycoparasite, protease

中图分类号:

S476

谢晓莉, 杨龙, 吴明德, 张静, 李国庆. 重寄生真菌盾壳霉胞外蛋白酶产生条件及酶活影响因子[J]. 中国生物防治学报, 2016, 32(3): 406-413.

XIE Xiaoli, YANG Long, WU Mingde, ZHANG Jing, LI Guoqing. Culture Condition and Characterization of Factors Affecting Activity of the Extracellular Proteases Produced by Mycoparasite Coniothyrium minitans[J]. journal1, 2016, 32(3): 406-413.

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重寄生真菌盾壳霉胞外蛋白酶产生条件及酶活影响因子

Culture Condition and Characterization of Factors Affecting Activity of the Extracellular Proteases Produced by Mycoparasite Coniothyrium minitans

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