Plasmid of pTRLI Transformation into Aspergillus terreus Protoplast with Polyethylenglycol

  • DUDI HARDIANTO BADAN PENGKAJIAN dan PENERAPAN TEKNOLOGI
  • MARLIA SINGGIH INSTITUTE OF TECHNOLOGY BANDUNG
  • AMIR MUSADAD INSTITUTE OF TECHNOLOGY BANDUNG
  • WAHONO SUMARYONO BADAN PENGKAJIAN dan PENERAPAN TEKNOLOGI
  • TUTUS GUSDINAR BADAN PENGKAJIAN dan PENERAPAN TEKNOLOGI

Abstract

Plasmid transformation is the introduction and incorporation of exogenous plasmid into cells or protoplast. The purpose of this research is transformation of pTRLI plasmid into protoplasts of Aspergillus terreus and obtain stable transformants. The research was initiated by isolation of pTRLI plasmid and determining its purity and concentration by nanodrop machine. Furthermore, protoplasts of A. terreus were isolated enzymatically by addition of chitinase, cellulase, and maserozyme enzymes. pTRLI plasmid was transformed into protoplasts of A. terreus by addition of calcium chloride and polyethyleneglycol (PEG) solutions. These transformants were grown in Czapek-Dox agar medium containing pyrithiamine 1 mg/L and the number of transformants/µg of pTRLI plasmid was calculated. The number of transformants were produced ranging from 12 to 19 transformants/µg of pTRLI plasmid. The success of the transformation was indicated by ptrA gene in transformants that could be amplified by PCR of 801 base pairs in size. It was concluded that PEG solution could be used to transform pTRLI plasmid into protoplasts of A. terreus and transformants are stable up to live generations by growing the transformants in Czapek-Dox agar medium containing pyrithiamine 1 mg/L.

References

1. Balajee MS. Aspergillus terreus complex, Medical Mycology. 2009.47:S42-S46.

2. Lai LT, Chih-Sheng Hung, and Chi-Chu Lo. Effect of Lactose and Glucose on Production of Itaconic Acid and Lovastatin by Aspergillus terreus ATCC 20542. J. Biosience and Bioengineering, 2007. 104(1):9-13.

3. Banuelos O, Leopoldo N, Javier C, Santiago G, and Juan F. Cotransformation with automous replicating and integrative plasmids in Penicillium chrysogenum in highly efficient and leads in some cases to of the intact integrative plasmid. Fungal Genetics and Biology. 2003. 40:83-92.

4. Barret V, Robert KD., and Paul AL. Genetic transformation of mycorrhiza fungus. Appl Microbiol Biotechnol. 1990. 33: 313-16.

5. Campoy S, Perez F, Martin IF, Gutierrez S, and Liras P. Stable Transformants of the Azaphilone PigmentProducting M onascus purpureus Obtained by Protoplast Transformation and Agrobacterium-mediated DNA Transfer Curr Genet. 2003. 43:447-52.

6. Mayer V. Genetic engineering of iilamentous fungiprogress, obstacles, and future trends. Biotechnol Adv. 2008. 26:177-85.

7. Herzog RW., Daniell H, Sigh NK, and PA Lemke. A Comparative Study on the Transformation of Aspergillus nidulans by Microprojectile Bombardment of Conidia and a More Conventional Procedur Using Protoplast Treated with Polyethylenglycol. Appl Microbiol. Biotechnol. 1996. 45:333-7.

8. Prabha VL. and NS Punekar. Genetic transformation in Aspergilli: tools of the trade. Indian J Biochem. Biophysics. 2004. 41:205-15.

9. Kubodera T., Nobuo Y, Akira N. Transformation of Aspergillus Sp. and Trichoderma reeesei using the pyrithiamine resistence gene (ptrA) of Aspergillus oryzae. Biosci Biotechnol Biochem. 2002. 66(2):404-6.

10. Lopez FJF, Elza F de Araujo, and Marisa V de Queiroz. Easy detection of green fluorescent protein multicopy transforrnants in Penicillium griseoreum. Gen and Molec Research. 2004. 3(4):449-55.

11. Skory CD, Perng-Kuang C, Jeff C, and John EL. Isolation and characterization of a gene from Aspergillus paratz'cus associated with the conversion of versicolorin A to sterigmatocystin in aflatoxin biosynthesis. App and Environ Microbiol. 1992. 3527-57.

12. Woloshuk CP, Seip ER, Payne GA, and Adkins CR. Genetic Transformation System for the AlflatoxinProducing Fungus Aspergillus flavus. Applied and Envirornental Microbiology. 1989. 55:86-90.

13. Gillian BT, Bradford C, J inyuan L, and Ning Z. Protoplast Transformation of Filamentous Fungi. lnzAmir Sharon, editors. Molecular and Cell Biology Method for Fungi. London: Humana Press. 2010. 3-19.
Published
2012-04-30
How to Cite
HARDIANTO, DUDI et al. Plasmid of pTRLI Transformation into Aspergillus terreus Protoplast with Polyethylenglycol. JURNAL ILMU KEFARMASIAN INDONESIA, [S.l.], v. 10, n. 1, p. 39-43, apr. 2012. ISSN 2614-6495. Available at: <http://jifi.farmasi.univpancasila.ac.id/index.php/jifi/article/view/275>. Date accessed: 24 nov. 2024.
Section
Articles