Preparation, regeneration and genetic system construction of Pochonia chlamydosporia protoplasts

doi:10.3969/j.issn.1004-1524.20240374

Acta Agriculturae Zhejiangensis ›› 2025, Vol. 37 ›› Issue (4): 800-807.DOI: 10.3969/j.issn.1004-1524.20240374

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S855.9

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.

Figures/Tables 9

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.

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

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

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

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

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

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.

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

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

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Preparation, regeneration and genetic system construction of Pochonia chlamydosporia protoplasts

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