RT-PCR analysis of in vivo expression of α-galactosidase gene in Phanerochaete chrysosporium ME446 grown on solid lignocellulosic substrate

M. S. Umi Kalsom

Abstract

Solid state fermentation of Phanerochaete chrysosporium ME446 was carried out using lignocellulose as substrate [mixture of ground wood pulp, wheat bran and sugar beet (PBS)]. Total RNA from solid cultures of P. chrysosporium ME446 was isolated using both conventional and RNeasy kit. High quality RNA was successfully isolated as shown by sharp bands (28S and 23S) on agarose gel electrophoresis. The level of transcript in mRNA population was then analysed by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR was also used to analyse the expression of
 α-galactosidase gene in P. chrysosporium ME446. Upon gel electrophoresis, the 260 bp fragment determined by AGEx1 and AGEx2 primers were observed. From RT-PCR analysis, the use of constitutively expressed trpC gene as a control experiment confirmed that, in all replicates of cultures, mRNA has been extracted and successfully used to make cDNA in comparable yields. In parallel with the isolation of RNA, α-galactosidase enzyme activities in the culture filtrate were determined.

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