中国兽医科学2025，Vol.55Issue(6)：780-788,9.DOI:10.16656/j.issn.1673-4696.2025.0112
基于正交试验的Pochonia chlamydosporia原生质体再生体系优化及生物学功能特性分析
Optimization of the protoplast regeneration system of Pochonia chlamydosporia based on orthogonal experiment and analysis of biological function characteristics
摘要
Abstract
This study aims to optimize the protoplast preparation and regeneration system of Pocho-nia chlamydosporia,laying a foundation for gene transformation,genetic modification,and biological function studies.In this study,the factors such as enzyme combinations,osmotic stabilizers,enzyme con-centrations,and enzymolysis time were systematically evaluated,optimizing the protoplast preparation and regeneration conditions.The results indicated that the optimal enzyme combination is cellulase+snail enzyme+lyticase,with an enzyme concentration of 1.5％,KCl as the osmotic stabilizer,and an enzy-molysis time of 4 hours,resulting in a protoplast yield of 3.95×106 protoplasts/mL.The best regeneration conditions were cellulase+lyticase combination,enzyme concentration of 2％,sorbitol,and an enzymoly-sis time of 1 hour,achieving a regeneration rate of 14.63％.Protoplast viability was confirmed using car-boxyfluorescein diacetate succinimidyl ester(CFDA-SE)fluorescence probe combined with laser confocal mi-croscopy.To assess the biological function stability of the regenerated strain,infection experiments were conducted to evaluate its infectivity to the eggs of three animal gastrointestinal nematodes.The results showed no significant difference in infectivity between the regenerated strain and the wild-type mycelium,indicating that the preparation and regeneration processes did not affect its biological function.This study successfully established a protoplast preparation and regeneration system for P.chlamydosporia,providing important insights for further exploring its molecular mechanisms in egg infection.关键词
卵寄生性真菌/原生质体制备/原生质体再生/生物防治Key words
egg-parasitic fungi/protoplast preparation/protoplast regeneration/biological control分类
农业科技引用本文复制引用
郝陆瑶,刘泓佑,马园,马成宇,李政忆,王瑞,王伟国..基于正交试验的Pochonia chlamydosporia原生质体再生体系优化及生物学功能特性分析[J].中国兽医科学,2025,55(6):780-788,9.基金项目
内蒙古自治区重点研发和成果转化计划项目(2023YFDZ0048) （2023YFDZ0048）
国家自然科学基金项目(32160838) （32160838）
国家乳业技术创新中心项目(2023-JSGG-5) （2023-JSGG-5）
鄂尔多斯市国家可持续发展议程创新示范区建设(含厅市会商、科技突围工程)科技支撑项目(KCX2024016) （含厅市会商、科技突围工程）
内蒙古自然科学基金项目(2023LHMS03022,2023LHMS03005,2021MS03088) （2023LHMS03022,2023LHMS03005,2021MS03088）
