| Fruiting-body formation is an important biological phenomena for edible fungus. Environmental conditions play a key role on formation of fruiting body. Vegetative mycelia are genetically able to fruit by various environmental factors, such as temperature, light, CO2 concentration, and biochemical stimuli. Temperature change has long been known to be necessary for fruit body formation in some species of edible fungus. In cultivation, fruiting is typically induced after vegetative growth by reducing the temperature for a certain amount of time. However, its molecular mechanism is unclear.Pleurotus pulmonarius belongs to fungi, Basidiomycetes, Agaricales, pleurotaceae, Pleurotus. " Nongxiu 1" is a new Pleurotus pulmonarius variety of heat tolerance,which selected by Zhejiang Academy of Agricultural Sciences through the cross breeding. Since the mature mycelia can be induced to form primordia by a sustained and transient cold stimulation, Nongxiu 1 is an ideal material for research on temperature response. In the previous study, a SSH (suppressive subtractive hybridization) cDNA library was constructed related to fruiting-body formation after cold stimulation. The result showed that this process not only related to the changes of the expression levels of genes related to downstream metabolic pathway and cell morphology and structure, but also related with the expression of the genes of the upstream global regulator. Transcription factors play a key role on this process, so it is necessary to clone and functionally analyze these transcription factors. In previous study, an EST (the expressed sequence tag) may encoding a CSL transcription factor (named PpCSL-1) was obtained from a SSH (suppressive subtractive hybridization) cDNA library related to this process. The previous reports showed that the CSL (CBF1/RBP-J?/Suppressor of Hairless/LAG-1) transcription factor family members play a critical role in cell division and fungal development.The full-length sequence of the candidate gene was obtained by TAIL PCR (thermal asymmetric interlaced PCR) based on the known sequences. And the complete cDNA sequence of Ppcsl-1 was cloned by 5' and 3' RACE (rapid-amplification of cDNA ends) contains 2991 bp. Ppcsl-1 has 4 introns and 3 exons. The PpCSL-1 contains three conserved domains including LAG1, BTD and IPT, and shows high evolutional conservation in DNA binding site. PpCSL-1 containing a hydrophobic structure located in BTD domain is a hydrophilic protein, which indicate that PpCSL-1 may be a transmembrane protein or a proteins bound to membrane. Thirty CSL sequence from fungi and metazoan were selected to construct a phylogenetic tree. Analysis of these CSL shows that the domain of CSL and the DNA binding sites were conservative in the evolutionary process. A phylogenetic analysis indicates that PpCSL-1 and PoCSL shared a highest homology among the CSLs from the basidiomycete fungi. To screen a suitable reference gene of real-time PCR analysis, six housekeeping genes of P.pulmonarius including ACT, GAPDH, eIF5, TUA, ATPase and RPL14 were selected to evaluate the expression stability by geNorm software. The result showed the RPL14 gene showed the most stable with a lowest M value of 0.496. RPL14 gene was selected to evaluate expression level of Ppcsl-1 gene in the developmental phases of P.pulmonarius. The result showed that Ppcsl-1 have highest expression level at the mycelia at 12 h by 5 ? cold stimulation across other developmental stages, which suggested that the Ppcsl-1 gene may be induced by cold stimulation and may play role on initiation of a series of developmental events of fruiting-body formation. |
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