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

参考文献 25

[1]	MANZANILLA-LÓPEZ R H, ESTEVES I, FINETTI-SIALER M M, et al. Pochonia chlamydosporia: advances and challenges to improve its performance as a biological control agent of sedentary endo-parasitic nematodes[J]. Journal of Nematology, 2013, 45(1): 1-7.
[2]	祝晓蕊, 许开华, 王新芳, 等. 一种食线虫性真菌: 厚垣普可尼亚菌(Pochonia chlamydosporia)ARSEF3539生长特性的研究[J]. 畜牧与饲料科学, 2017, 38(4): 1-4.
	ZHU X R, XU K H, WANG X F, et al. Growth characteristics of a nematode-trapping fungus: Pochonia chlamydosporia ARSEF3539[J]. Animal Husbandry and Feed Science, 2017, 38(4): 1-4. (in Chinese with English abstract)
[3]	樊雅茹. 厚垣普可尼亚菌与埃普利诺菌素联合制剂的研究[D]. 呼和浩特: 内蒙古农业大学, 2021.
	FAN Y R. Study on the combination preparation of Pochonia chlamydosporia and eprinomection[D]. Hohhot: Inner Mongolia Agricultural University, 2021. (in Chinese with English abstract)
[4]	张艳妮, 郝陆瑶, 马园, 等. 厚垣普可尼亚菌ARSEF9494菌株最适碳源氮源碳氮质量比及初始pH值的筛选[J]. 中国兽医科学, 2023, 53(12): 1544-1553.
	ZHANG Y N, HAO L Y, MA Y, et al. Screening on the optimal source of carbon and nitrogen, carbon-nitrogen mass ratio, and initial pH value for Pochonia chlamydosporia ARSEF9494 in solid and liquid media[J]. Chinese Veterinary Science, 2023, 53(12): 1544-1553. (in Chinese with English abstract)
[5]	CASTRO L S, MARTINS I, ALVES V M, et al. Effect of the Enzymatic Fungal Extract of Pochonia chlamydosporia on the Viability of Fasciola hepatica eggs[J]. Journal of Advanced Veterinary Research, 2020, 10(3): 135-140.
[6]	DIAS A S, ARAÚJO J V, BRAGA F R, et al. Pochonia chlamydosporia in the biological control of Fasciola hepatica in cattle in Southeastern Brazil[J]. Parasitology Research, 2013, 112(6): 2131-2136.
[7]	Araújo JV, Braga FR, Silva AR, et al. In vitro evaluation of the effect of the nematophagous fungi Duddingtonia flagrans, Monacrosporium sinense, and Pochonia chlamydosporia on Ascaris suum eggs[J]. Parasitology Research, 2008 102(4):787-790.
[8]	ARAUJO J M, DE ARAÚJO J V, BRAGA F R, et al. Activity of the nematophagous fungi Pochonia chlamydosporia, Duddingtonia flagrans and Monacrosporium thaumasium on egg capsules of Dipylidium caninum[J]. Veterinary Parasitology, 2009, 166(1/2): 86-89.
[9]	郝陆瑶, 焦万明, 尹衍峰, 等. 厚垣普可尼亚菌发酵液中胞外酶的活性分析及其杀虫毒力测定[J]. 中国兽医科学, 2023, 53(9): 1158-1165.
	HAO L Y, JIAO W M, YIN Y F, et al. Analysis of extracellular hydrolyase activity in Pochonia chlamydosporia fermentation broth and its determination of nematode killing virulence[J]. Chinese Veterinary Science, 2023, 53(9): 1158-1165. (in Chinese with English abstract)
[10]	姚玉荣, 霍建飞, 郝永娟, 等. 根结线虫生防真菌交枝顶孢原生质体的制备与再生体系构建[J]. 植物保护, 2020, 46(6): 149-154.
	YAO Y R, HUO J F, HAO Y J, et al. Preparation and regeneration of protoplasts from the biocontrol fungus Acremonium implicatum[J]. Plant Protection, 2020, 46(6): 149-154. (in Chinese with English abstract)
[11]	沈慧敏, 李超, 高利, 等. 原生质体法介导真菌遗传转化的研究进展[J]. 植物保护, 2017, 43(2): 25-28.
	SHEN H M, LI C, GAO L, et al. Research progress in transformation of fungi mediated by protoplasts[J]. Plant Protection, 2017, 43(2): 25-28. (in Chinese with English abstract)
[12]	赵美, 王陈骄子, 周而勋, 等. 丝状真菌遗传转化系统的最新研究进展[J]. 基因组学与应用生物学, 2019, 38(3): 1138-1143.
	ZHAO M, WANG C, ZHOU E X, et al. The latest research progress on genetic transformation system of filamentous fungi[J]. Genomics and Applied Biology, 2019, 38(3): 1138-1143. (in Chinese with English abstract)
[13]	沈宝明. 厚垣孢普可尼亚菌线虫卵寄生相关胞外蛋白酶功能研究[D]. 长沙: 湖南农业大学, 2015.
	SHEN B M. The functional research of extracellular proteases related to nematode egg parasitism in Pochonia chlamydosporia[D]. Changsha: Hunan Agricultural University, 2015. (in Chinese with English abstract)
[14]	刘璐, 高原, 杜双田, 等. 香菇YJ-01原生质体制备与再生条件的优化[J]. 食用菌, 2017, 39(5): 16-23.
	LIU L, GAO Y, DU S T, et al. Optimization of protoplast preparation and regeneration conditions of Lentinula edodes YJ-01[J]. Edible Fungi, 2017, 39(5): 16-23. (in Chinese)
[15]	庄霞. 厚垣普可尼亚菌PC-170诱导植物系统抗性作用机理及其在葡萄上应用研究[D]. 长沙: 湖南农业大学, 2021.
	ZHUANG X. The mechanism research of Pochonia chlamydosporia PC-170 in inducing plant systemic resistance and its application in grapes[D]. Changsha: Hunan Agricultural University, 2021. (in Chinese with English abstract)
[16]	张晓妍, 谢昭, 王秀华, 等. 人参灰葡萄孢原生质体制备条件的优化及再生菌株致病性研究[J]. 植物病理学报, 2024, 54(1): 160-169.
	ZHANG X Y, XIE Z, WANG X H, et al. Condition optimization for protoplast preparation of Botrytis cinerea and the pathogenicity of regenerated strains[J]. Acta Phytopathologica Sinica, 2024, 54(1): 160-169. (in Chinese with English abstract)
[17]	郭爱华. 非洲紫罗兰原生质体制备条件优化[J]. 江苏农业科学, 2015, 43(10): 230-232.
	GUO A H. Optimization of African violet protoplast preparation conditions[J]. Jiangsu Agricultural Sciences, 2015, 43(10): 230-232. (in Chinese)
[18]	张伟, 杨晓野, 王瑞, 等. 捕食性真菌Duddingtonia flagrans原生质体获得与再生的荧光标记评价[J]. 菌物学报, 2017, 36(3): 302-310.
	ZHANG W, YANG X Y, WANG R, et al. Fluorescence labeling of production and regeneration of protoplasts from the nematode-trapping fungus Duddingtonia flagrans[J]. Mycosystema, 2017, 36(3): 302-310. (in Chinese with English abstract)
[19]	邹优花, 连玲丹, 钟武杰, 等. 杏鲍菇原生质体制备、再生及其遗传转化体系的建立[J]. 食品研究与开发, 2023, 44(21): 138-144.
	ZOU Y H, LIAN L D, ZHONG W J, et al. Preparation and regeneration of protoplasts of Pleurotus eryngii and establishment of its genetic transformation system[J]. Food Research and Development, 2023, 44(21): 138-144. (in Chinese with English abstract)
[20]	高成昱, 王艺衡, 靳江周, 等. 梨叶片原生质体制备方法的建立及其基因瞬时转化试验[J]. 园艺学报, 2023, 50(5): 1141-1150.
	GAO C Y, WANG Y H, JIN J Z, et al. Method for high efficiency isolation of protoplasts from pear leaves and its utility for gene transient expression[J]. Acta Horticulturae Sinica, 2023, 50(5): 1141-1150. (in Chinese with English abstract)
[21]	赵文超, 牛延宁, 金明飞, 等. 茂源链霉菌原生质体的制备与再生[J]. 西北农林科技大学学报(自然科学版), 2015, 43(11): 187-192.
	ZHAO W C, NIU Y N, JIN M F, et al. Preparation and regeneration of protoplast from Streptoverticillium mobaraensis[J]. Journal of Northwest A & F University(Natural Science Edition), 2015, 43(11): 187-192. (in Chinese with English abstract)
[22]	赵小强, 陈志荣, 何芳, 等. 大丽轮枝菌原生质体的制备及再生[J]. 生物技术通报, 2018, 34(7): 166-173.
	ZHAO X Q, CHEN Z R, HE F, et al. Preparation and regeneration of protoplast from Verticillium dahliae[J]. Biotechnology Bulletin, 2018, 34(7): 166-173. (in Chinese with English abstract)
[23]	MA W J, YI F, XIAO Y, et al. Isolation of leaf mesophyll protoplasts optimized by orthogonal design for transient gene expression in Catalpa bungei[J]. Scientia Horticulturae, 2020, 274: 109684.
[24]	龚伟杰, 刘洪, 黄萍, 等. 番茄匍柄霉原生质体的制备与再生体系构建[J]. 浙江农业学报, 2022, 34(2): 337-342.
	GONG W J, LIU H, HUANG P, et al. Optimization of protoplast preparation and regeneration conditions of Stemphylium lycopersici[J]. Acta Agriculturae Zhejiangensis, 2022, 34(2): 337-342. (in Chinese with English abstract)
[25]	赵楠, 闫思远, 裴瑞瑞, 等. 深色有隔内生真菌S12菌株遗传转化体系的建立及GFP标记菌株的获得[J]. 西北农林科技大学学报(自然科学版), 2024, 52(4): 127-135.
	ZHAO N, YAN S Y, PEI R R, et al. PEG-mediated transformation system of dark septate endophyte Cladosporium cladosporioides S12 with GFP gene for strain visualization[J]. Journal of Northwest A & F University(Natural Science Edition), 2024, 52(4): 127-135. (in Chinese with English abstract)

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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