Optimization of theprotoplast electrotransformation system and analysis of biolog-ical functions of Pochonia chlamydosporia based on orthogonal experiments
10.16303/j.cnki.1005-4545.2025.11.16
- VernacularTitle:基于正交试验的Pochonia chlamydosporia原生质体电转化体系优化及生物学功能分析
- Author:
Luyao HAO
1
;
Hongyou LIU
;
Fengmiao ZHAO
;
Yuan MA
;
Chengyu MA
;
Zhengyi LI
;
Rui WANG
Author Information
1. 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
- Publication Type:Journal Article
- Keywords:
egg-parasitic fungi;
protoplast transformation;
electroporation;
biological function;
bio-logical control
- From:
Chinese Journal of Veterinary Science
2025;45(11):2420-2429,2456
- CountryChina
- Language:Chinese
-
Abstract:
This study aims to establish an efficient protoplast-mediated genetic transformation sys-tem for Pochonia chlamydosporia(P.chlamydosporia)to facilitate gene transformation,genetic modification,and subsequent biological function research.Through orthogonal testing,the study systematically optimized key factors influencing electroporation,including voltage,osmotic stabi-lizer,pulse time,nucleic acid concentration,and protoplast concentration.The results showed that the optimal electroporation conditions were:voltage of 250 V,osmotic stabilizer as 0.6 mol/L su-crose solution,pulse time of 10 ms,plasmid concentration of 1％,and protoplast concentration of 1×107 protoplasts/mL,yielding a transformation efficiency of 0.4 × 103 CFU/pg.The interaction analysis revealed that the five factors affected transformation efficiency in the following order:plasmid concentration＞protoplast concentration＞pulse time＞osmotic stabilizer type＞voltage.Based on the positive transformants,the study further evaluated their colony morphology,growth rate,conidial yield,mycelial dry weight,and the ability to infect the eggs of three types of animal gastrointestinal nematodes.The results indicated that the electroporation process did not signifi-cantly affect the biological functions of P.chlamydosporia.The transformants successfully ex-pressed enhanced green fluorescent protein(EGFP),and PCR analysis confirmed the successful in-tegration of the thiostrepton resistance gene.In conclusion,this study successfully established a protoplast-based electroporation-mediated genetic transformation system for P.chlamydosporia,providing a valuable foundation for further research into the mechanism of egg parasitism and the underlying genes involved in its biocontrol activity.
