| Carbon catabolite repression (CCR) is a phenomenon of which while preferred carbon sources are present, the expression of enzymes functioning during the degradation of difficult carbon sources is repressed. CCR is the main restrictive factor in the production of cellulase.Protein phosphorylation is one of the most important mechanisms for the post-translational modification, and plays an important role in eukaryotic cells. In eukaryotic organisms, casein kinase Ⅱ(CKⅡ) is a highly conserved serine/threonine protein kinase with a growing list of more than hundreds of substrates. The majority of substrates are proteins implicated in signal transduction, gene expression and other nuclear functions.Trichoderma reesei (teleomorph Hypocrea jecorina) is the important model cellulolytic fungus with strong glycoside hydrolase activities. Reports suggest Crel of T. reesei may be phosphorylated by a casein kinase Ⅱ-like. CK Ⅱ may be related to metabolism of carbon source.In this paper.we focus on the biology function of CK Ⅱ in T. reesei.In T. reesei, CKⅡis a heterotetramer composed of two catalytic subunits (α1 or α2) and two regulatory subunits(β1 or β2), the catalytic subunits have catalytic activity. In order to study the biology function of CK Ⅱ in T. reesei, we try to knock out the catalytic subunits using gene targeting technique. We obtained the catalytic subunit gene α2 knock out mutant △α2. However, the catalytic subunit gene al can’t completely knock out only existing in heterozygote, therefore we presume that the catalytic subunit gene α1 plays a pivotal role on growth and development in T. reesei. It has not been successful to disturb α1 using RNA interference.We analyzd the mycelium phenotype of wild type strain △tku70 and knockout mutant strain △α2 in glucose as a single carbon source of liquid medium.The mycelium of △tku70 in liquid medium is a small ball, but the mycelium of △α2 spreads, and mycelia winding and balling obviously weakened.Meanwhile, compered to wild type strain △tku70, the hyphae bifurcation number of △α2 obviously decreases. By using ImageJ software, the results show that the deletion of α2 caused 50% increased of the average distance of hyphae bifurcation, and 15% decreased of the second hyphae length from the top.We analyzed transcript level of 11 chitinases.Compered to wild type strain △tku70,5 chitinases of △α2 have signigicantly decreased,5 chitinases of △α2 have little change, only 1 chitinase have slightly increased.The results show that the deletion of α2 leads to most of transcript level of chitinases signigicantly decrease. Chitin is a major component of fungal cell wall. The distance of hyphae bifurcation will become longer and the hyphae length from the top will become shorter due of transcript level of chitinases decrease.We analyzed spore production of △tku70 and △α2, the deletion of a2 caused 73% increased of spore production.We analyzed the colony area of wild type strain △tku70 and gene knockout mutants △α2 in glucose as a single carbon source of solid medium, the deletion of a2 caused 18% increased of colony area. It has the extremely significant difference by the statistical tests.We analyzed the glucose concentration and biomass of wild type strain △tku70 and gene knockout mutants △α2 by submerged liquid fermentation of glucose as carbon source. The results show that growth speed and glucose consumption rete of △α2 obviously higher than wild type strain △tku70. The results show that CKⅡhas a negative regulation on the spore production and mycelia growth.In order to study the influence of CKⅡ on cellulase, we measured cellulase activities, extracelluar protein, transcription level of cellulases and transcription factors.The results show that the regulatory mechanism of CKⅡ on cellulase is complex, which relates to different carbon source. At the same time, cellulase activities and extracelluar prote between △α2 and △tku70 have little change may be due to the synergistic effect of celulases and transcription factors. |
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