周世萍,,
杨广斌,
解思达,
朱鑫泽
西南地区林业生物质资源高效利用国家林业与草原局重点实验室(西南林业大学), 昆明 650224
作者简介: 熊张平(1995-),男,硕士研究生,研究方向为生态毒理学,E-mail:xzp521821@dingtalk.com.
通讯作者: 周世萍,kmzhoushiping@163.com ;
基金项目: 国家自然科学基金资助项目（31860155）；云南省农业联合面上项目（2017FG001（-040））；西南地区林业生物质资源高效利用国家林业和草原局重点实验室开放基金（2019-KF13）中图分类号: X171.5
Earthworm Acrosome Effect and Its Application in Soil Health Quality Diagnosis
Xiong Zhangping,Zhou Shiping,,
Yang Guangbin,
Xie Sida,
Zu Xinze
Key Laboratory of State Forestry Administration for Highly-Efficient Utilization of Forestry Biomass Resources in Southwest China(Southwest Forestry University), Kunming 650224, China
Corresponding author: Zhou Shiping,kmzhoushiping@163.com ;
CLC number: X171.5
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摘要:蚯蚓在评价土壤生态环境污染方面起着重要作用，但目前与生殖毒性相关的蚯蚓生物标志物中缺乏可以灵敏和快速对污染物生殖毒性进行诊断的生物标志物。本研究采用考马斯亮蓝法对蚯蚓精子顶体反应进行检测；采用响应面分析法优化苯甲酰精氨酸乙酯/乙醇脱氢酶（BAEE/ADH）联合测定法，以建立蚯蚓精子顶体酶活性测定法。结果表明，蚯蚓精子顶体率为（47.67±3.87）%，最优测定条件为洗涤2次，抽提时间24 h，ADH浓度1.8 mg·mL-1，氧化型辅酶Ⅰ（NAD）浓度12 mg·mL-1。为考察方法的可靠性，采用醋酸棉酚污染土壤暴露实验进行方法学考察。数据表明，醋酸棉酚污染土壤暴露3 d后，检测出顶体酶活性变化，且与精子密度具有一定的相关性（r=0.9908）。建立的蚯蚓顶体酶活性测定方法可以快速和有效的检测醋酸棉酚的生殖毒性变化，蚯蚓顶体酶活性有望作为生物标志物应用于土壤污染的生态毒理学快速诊断中。
关键词: 醋酸棉酚/
蚯蚓/
顶体反应/
顶体酶活性/
生物标志物
Abstract:Earthworm plays an important role in the evaluation of soil pollution, but currently there is a lack of earthworm biomarkers that could sensitively and quickly diagnose the reproductive toxicity of pollutants. In this study, the acrosomal reaction of earthworm sperm was detected by Coomassie Bright Blue Method. In order to establish a method for the determination of acrosomal activity of earthworm sperm, Response Surface Methodology was used to optimize the combined determination of benzoyl arginine ethyl ester/ethanol dehydrogenase (BAEE/ADH). The results showed that the acrosomal rate of earthworm sperm was (47.67±3.87)%. The optimal condition was washed for 2 times, extracted for 24 h, ADH concentration as 1.8 mg·mL-1, and oxidized coenzyme Ⅰ (NAD) concentration as 12 mg·mL-1. To investigate the reliability of the method, gossypol acetate soil exposure test was used to investigate the methodology. The results showed that acrosomal enzyme activity changed and correlated with sperm density (r=0.9908) in the soil exposed to gossypol acetate for 3 d. The established method for the determination of earthworm acrosomal activity could quickly and effectively detect the reproductive toxicity of gossypol acetate. The earthworm acrosomal enzyme activity is expected to be used as a biomarker for the rapid ecotoxicological diagnosis of soil pollution.
Key words:gossypol acetate/
the earthworm/
acrosomal reaction/
acrosomal enzyme activity/
biomarker.
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