厚垣普可尼亚菌原生质体的制备、再生与遗传体系构建

doi:10.3969/j.issn.1004-1524.20240374

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (4): 800-807.DOI: 10.3969/j.issn.1004-1524.20240374

厚垣普可尼亚菌原生质体的制备、再生与遗传体系构建

马园(), 郝陆瑶, 张艳妮, 李倩楠, 王瑞^*()

内蒙古农业大学兽医学院,农业农村部动物疾病临床诊疗技术重点实验室,国家级动物医学实验教学中心,内蒙古呼和浩特 010011

收稿日期:2024-04-26 出版日期:2025-04-25 发布日期:2025-05-09
作者简介:马园(1995—),女,内蒙古乌海人,博士研究生,研究方向为寄生虫病及其防治。E-mail:mayuan0820@126.com
通讯作者: *王瑞,E-mail:wr2006@163.com
基金资助:
国家自然科学基金(31960724);国家自然科学基金(32160838);国家自然科学基金(31460656);内蒙古自治区重点研发和成果转化计划项目(2023YFDZ0048);内蒙古自然科学基金项目(2023LHMS03022);内蒙古自然科学基金项目(2023LHMS03005);内蒙古自然科学基金项目(2021MS03088);内蒙古自然科学基金项目(2019MS03017);内蒙古自治区高等学校科学技术研究项目(NJZY21495)

Preparation, regeneration and genetic system construction of Pochonia chlamydosporia protoplasts

MA Yuan(), HAO Luyao, ZHANG Yanni, Ll Qiannan, WANG Rui^*()

Key Laboratory of Clinical Diagnosis and Treatment of Anima Diseases, Ministry of Agriculture and Rural Affairs, National Animal Medicine Experimental Teaching Center, College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010011, China

Received:2024-04-26 Online:2025-04-25 Published:2025-05-09

摘要/Abstract

摘要： 为建立厚垣普可尼亚菌高效原生质体制备体系和聚乙二醇(PEG)介导的遗传转化体系,选取厚垣普可尼亚菌菌丝作为实验对象,系统考察菌丝培养时间、裂解酶种类、酶解时间与温度、渗透压稳定剂类型和pH值等关键参数对原生质体形成的影响;采用单因素试验优化原生质体制备工艺;通过PEG介导法进行质粒DNA转化,评价转化子的遗传稳定性。结果表明:厚垣普可尼亚菌原生质体的最佳制备条件为厚垣普可尼亚菌在马铃薯葡萄糖水(PDB)培养基中26 ℃培养24 h,使用0.7 mol·L^-1 NaCl(pH值7.5)作为渗透压稳定剂,经20 mg·mL^-1崩溃酶在30 ℃酶解5 h,0.5 g菌丝的原生质体产量达3.05×10⁶ mL^-1;获得的原生质体在再生培养基中的再生率为6.2%。PEG介导转化能够获得稳定遗传的转化子,转化率为45%,转化菌株的菌落形态、生长速率和分生孢子产量与野生菌株无显著差异。本研究成功建立了厚垣普可尼亚菌原生质体高效制备与遗传转化体系,为其生防功能解析和基因工程学改造提供了关键技术支撑。

关键词: 厚垣普可尼亚菌, 原生质体, 酶解条件, 遗传转化

Abstract:

To establish an efficient protoplast preparation system and PEG-mediated genetic transformation system for Pochonia chlamydosporia, mycelial culture time, lysing enzyme types, enzymatic hydrolysis time/temperature, osmotic stabilizer type and its pH value were systematically investigated. Protoplast preparation was optimized through single-factor experiments. Plasmid DNA transformation was performed via PEG-mediated method, with genetic stability of transformants evaluated. The results showed that optimal preparation conditions were 24-h-old mycelia cultured in PDB medium at 26 ℃, treated with 20 mg·mL^-1 lysing enzymes (driselase) in 0.7 mol·L^-1 NaCl (pH 7.5) at 30 ℃ for 5 h, which yielded 3.05×10⁶ mL^-1 protoplasts from 0.5 g mycelia. Protoplast regeneration rate reached 6.2% on regeneration medium. PEG-mediated transformation achieved 45% efficiency with stable inheritance. Transformants showed no significant differences from the wild strain in colony morphology, growth rate and conidium yield. This study successfully established efficient protoplast preparation and genetic transformation system for P. chlamydosporia, providing crucial technical support for functional genomics research and genetic engineering applications in biocontrol mechanisms.

Key words: Pochonia chlamydosporia, protoplast, enzymatic condition, genetic transformation

中图分类号:

S855.9

马园, 郝陆瑶, 张艳妮, 李倩楠, 王瑞. 厚垣普可尼亚菌原生质体的制备、再生与遗传体系构建[J]. 浙江农业学报, 2025, 37(4): 800-807.

MA Yuan, HAO Luyao, ZHANG Yanni, Ll Qiannan, WANG Rui. Preparation, regeneration and genetic system construction of Pochonia chlamydosporia protoplasts[J]. Acta Agriculturae Zhejiangensis, 2025, 37(4): 800-807.

图/表 9

图1 菌丝培养时间对厚垣普可尼亚菌原生质体产量的影响柱上无相同小写字母表示差异显著(P<0.05)。下同。

Fig.1 Influence of mycelial culture time on the yield of P. chlamydosporia protoplasts Bars marked without the same lowercase letter indicates significant differences at P<0.05. The same as below.

图2 裂解酶对厚垣普可尼亚菌原生质体产量的影响

Fig.2 Effect of lysing enzymes on the yield of P. chlamydosporia protoplasts

图3 酶解温度和时间对厚垣普可尼亚菌原生质体产量的影响

Fig.3 Effect of enzymatic hydrolysis temperature/time on the yield of P. chlamydosporia protoplasts

图4 渗透压稳定剂和pH值对厚垣普可尼亚菌原生质体产量的影响

Fig.4 Effect of osmotic stabilizers and pH value on the preparation of protoplasmic systems of P. chlamydosporia

图5 厚垣普可尼亚菌原生质体(箭头所示)(A)和再生菌丝(B)

Fig.5 Protoplasts (shown by arrows) (A) and regenerating mycelium (B) of P. chlamydosporia

图6 转化子的PCR鉴定结果 M,DL2 000 DNA marker;1~11,转化子;12,阳性对照;13,阴性对照。

Fig.6 The results of PCR identification of transformants M, DL2 000 DNA marker; 1-11, Transformants; 12, Positive control; 13, Negative control.

图7 厚垣普可尼亚菌转化子与野生菌株的菌落形态 A,转化子菌落正面;B,转化子菌落背面;C,野生菌株菌落正面;D,野生菌株菌落背面。

Fig.7 Colony morphology of transformants and wild strain of P. chlamydosporia A, Front of transformant colony; B, Back of transformant colony; C, Front side of wild strain colony; D, Back side of wild strain colony.

图8 厚垣普可尼亚菌转化子(A)和野生菌株(B)的菌丝形态图

Fig.8 Mycelial morphology of transformants(A) and wild strain (B) of P. chlamydosporia

图9 厚垣普可尼亚菌野生菌株与转化子的菌丝的生长情况

Fig.9 Mycelial growth of wild strain and transformants of P. chlamydosporia

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厚垣普可尼亚菌原生质体的制备、再生与遗传体系构建

Preparation, regeneration and genetic system construction of Pochonia chlamydosporia protoplasts

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