CN105543212A - 真核微生物抗苯菌灵标记突变株的选育方法 - Google Patents
真核微生物抗苯菌灵标记突变株的选育方法 Download PDFInfo
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Abstract
本发明涉及一种真核微生物抗苯菌灵标记突变株的选育方法，针对真核微生物具体采用紫外诱变方法获得抗苯菌灵标记的突变株。通过本发明方法可以获得抗50μg/mL苯菌灵突变株。本发明所述的抗苯菌灵突变菌株的选育方法，可广泛应用于青霉属等真核微生物抗性标记的筛选。
Description
技术领域
本发明属于诱变育种中筛选标记选育领域，涉及真核微生物经紫外诱变产生抗苯菌灵突变株的诱变方法。
背景技术
真核微生物代谢产物已广泛应用于我们生活和生产中，为了进一步提高其产量，获得具有抗性标记突变株是选育高产突变株的关键。
研究表明，原始真核微生物是不具有抗苯菌灵的特性，同时原始真核微生物生产代谢产物的水平普遍偏低，而基因组重排是真核微生物改造的主要方法，但是首要条件是要获得具有抗苯菌灵标记的出发菌株。
发明内容
为了克服现有技术存在的上述问题，本发明提供了一种基于紫外诱变技术用于筛选抗苯菌灵突变株的方法，对比其它物理和化学诱变方法，本发明方法简便实用，特别适用于青霉属等真核微生物抗苯菌灵标记的筛选。
本发明提出了一种真核微生物抗苯菌灵标记突变株的选育方法，所述紫外诱变程序：
距紫外线距离，10-30cm；
诱变时间，40-120s；
本发明还提出了一种真核微生物抗苯菌灵标记突变株的选育方法，其特征在于孢子制备、初筛和复筛，所述方法包括以下步骤：
步骤一、真核微生物孢子菌悬液的制备，其包括：将真核微生物接种于PDA培养基中28℃培养5-7天，用50mL去离子水洗脱孢子，将孢子放入装有已灭菌玻璃珠的三角瓶中，于200rpm、28℃摇20min，再用已灭菌的含有棉花的漏斗过滤除去菌丝体和培养基，获得单孢子悬浮液；
步骤二、将获得的单孢子悬浮液距紫外线距离10-30cm，诱变时间为40-120s获得突变株；
步骤三、取100μL的稀释菌液涂布于初筛选培养基(含50μg/mL苯菌灵)上，初筛获得抗苯菌灵的突变株；
步骤四、将步骤三获得的突变株传接3代于含有50μg/mL苯菌灵的初筛培养基上，防止回复突变。
本发明在上述整个操作过程中，可以一次获得含有抗苯菌灵的突变株，大大降低以往反复繁重的筛选强度；
综上所述，本发明提供了真核微生物抗苯菌灵的诱变方法，为真核微生物抗性诱变提供了快捷适用的技术。
具体实施方式
通过以下具体实施例，对本发明作详细说明，本发明的保护内容不局限于以下实施例。本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。实施本发明的过程、条件、试剂、实验方法等，除本领域的普遍知识和公知常识外，本发明没有特别限制内容。
1、草酸青霉16孢子的紫外诱变程序：
距紫外线距离，20cm；
诱变时间，60s。
2、草酸青霉16抗苯菌灵标记突变株的选育方法，所述方法包括以下步骤：
草酸青霉16孢子菌悬液的制备，其包括：将草酸青霉16接种于PDA培养基中28℃培养7天，用50mL去离子水洗脱孢子，将孢子放入装有已灭菌玻璃珠的三角瓶中，于200rpm、28℃摇20min，再用已灭菌的含有棉花的漏斗过滤除去菌丝体和培养基，获得单孢子悬浮液；
用上述草酸青霉16孢子的紫外诱变程序处理单孢子悬浮液获得突变株；
取100μL的稀释菌液涂布于初筛选培养基(含50μg/mL苯菌灵)上，初筛获得抗苯菌灵的突变株；
初筛培养基：1-2％溶胀纤维素，0.3％KH2PO4，0.2％(NH4)2SO4，0.05％尿素，0.05％MgSO4，0.05％CaCl2，1‰(v/v)孟德尔盐溶液，50μg/mL苯菌灵，1.6-2％琼脂粉，根据水解圈大小来筛选；
复筛并验证抗苯菌灵突变株，将初筛的突变株接种于复筛培养基中，每个突变株复筛3次，防止回复突变；
复筛培养基包括：1-2％溶胀纤维素，0.3％KH2PO4，0.2％(NH4)2SO4，0.05％尿素，0.05％MgSO4，0.05％CaCl2，1‰(v/v)孟德尔盐溶液，50μg/mL苯菌灵，通过DNS方法测定酶活。
Claims (1)
1.一种真核微生物抗苯菌灵标记突变株的选育方法，其特征在于：先将真核微生物孢子紫外诱变，紫外诱变程序为距紫外线距离10-30cm、诱变时间，40-120s；选育方法为：
步骤一、真核微生物孢子菌悬液的制备，其包括：将真核微生物接种于PDA培养基中28℃培养5-7天，用50mL去离子水洗脱孢子，将孢子放入装有已灭菌玻璃珠的三角瓶中，于200rpm、28℃摇20min，再用已灭菌的含有棉花的漏斗过滤除去菌丝体和培养基，获得单孢子悬浮液；
步骤二、将获得的单孢子悬浮液距紫外线距离10-30cm，诱变时间为40-120s获得突变株；
步骤三、取100μL的稀释菌液涂布于初筛选培养基(含50μg/mL苯菌灵)上，初筛获得抗苯菌灵的突变株；
步骤四、将步骤三获得的突变株传接3代于含有50μg/mL苯菌灵的初筛培养基上，防止回复突变。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104342379A (zh) * | 2013-07-23 | 2015-02-11 | 江苏省微生物研究所有限责任公司 | 一种利用磷霉素选育达托霉素高产菌株的方法 |
| CN104774834A (zh) * | 2015-04-14 | 2015-07-15 | 中国医药集团总公司四川抗菌素工业研究所 | 采用谷氨酸钠耐受性模型筛选达托霉素高产菌株的方法 |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104342379A (zh) * | 2013-07-23 | 2015-02-11 | 江苏省微生物研究所有限责任公司 | 一种利用磷霉素选育达托霉素高产菌株的方法 |
| CN104774834A (zh) * | 2015-04-14 | 2015-07-15 | 中国医药集团总公司四川抗菌素工业研究所 | 采用谷氨酸钠耐受性模型筛选达托霉素高产菌株的方法 |
Non-Patent Citations (3)
| Title |
|---|
| G.C.PAPAVIZAS ET AL: "Physiological and Biocontrol Characteristics of Stable Mutants of Trichoderma viride Resistant to MBC Fungicides", 《PHYTOPATHOLOGY》 * |
| 乔长晟等: "基于核糖体工程理论的常压室温等离子体诱变筛选多杀菌素高产菌", 《中国生物工程杂志》 * |
| 杨合同等: "绿色木霉LTR-2菌株的紫外线诱变改良", 《中国生物防治》 * |
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