Tecnicas de protoplastização e isolamento de DNA de alto peso molecular de aspergillus niger

AUTOR(ES)
DATA DE PUBLICAÇÃO

1987

RESUMO

This research was done aiming the improvement of two methods of great relevanceto the Recombinant DNA Tech-nology (RDT) in Aspergillus niger. Firstly, the method of production and regeneration of protoplasts was improved. The best protoplasts preparations (2. 1 x 108 protoplasts/ml) were obtained when the my-celium was grown in medium containing Tween 80 and magne-sium sulphate 0.5M, the latter used as an osmotic stabili-zer. However, regeneration could only be significantly im-proved when magnesium sulphate was used at a lower concen-tration (50 mM) associated with sorbitol (1.2 M) during the production of protoplasts. Cytological analysis with protoplasts isolated by using the method of GAMBINO et alii (1984)revealed that up to 32 nuclei can be counted per protoplast. The method for isolation of DNA from this species was also improved. The final procedure is rapid (3.5 hours of manipulation before cesium chloride ? ethidium bromide gradient) and DNA isolated has showed the following characteristics: - high molecular weight; approximately 50 Kb. ? low level of proteic contamination (A260: A280= 1,8 - 2,0).- susceptibility to restriction (Eco RI and Pst I) and ligation (T4 DNA - ligase) enzymes. The DFA method was -.applied to estimate the recovery of DNA by using this improved procedure. DNA yields as high as 70% (ratio between final and inicial DNA concentrations) were obtained. The improved procedure has also proved to be suita-ble for extraction of DNA from others species of filamen-tous fungi.

ASSUNTO(S)

protoplastos acido desoxirribonucleico

ACESSO AO ARTIGO

Documentos Relacionados