Studies On The Gene Expression Of Different Metabolic Phase Of Phanerochaete Chrysosporium

The white-rot basidiomycete-Phanerochaete chrysosporium is an excellent model organism for studying the mechanism of lignin biodegradation and its genomic sequence has been published by Joint Genome Institute of USA. P. chrysosporium can produce two kinds of H₂O₂-dependent peroxidase isozymes, lignin peroxidase (LiP) and manganese peroxidase (MnP), which are major components in lignin degradation. The previous investigations showed that both of them were produced during the secondary metabolism, and their production was greatly influenced by various factors, such as various nutrition factors and oxygen. Molecular biology study revealed that lignin and manganese peroxidase are encoded by genes in two gene families, respectively. The transcription of these genes is regulated by nitrogen, carbon, Mn²⁺, molecular O₂ and heat shock, etc. However, the regulatory mechanism of these genes was not clear during the secondary metabolism. Therefore, it was very important to explain the mechanism of transcriptional regulation of these genes both in theory and practice. Studies on the mechanism of transcriptional regulation of the P. chrysosporium during the secondary metabolism were carried out in the following 4 aspects and corresponding results were obtained.First, the study was initiated to identify phase-specific expression genes of P. chrysosporium mycelial growth under nitrogen-limited conditions at 48h and 72h incubation time. Two. suppression subtractive hybridization (SSH) libraries were constructed that is 72h-specific library containing 1728 cDNA clones and 48h-specific one with 868 cDNA clones. PCR was used to amplify cDNA fragment in each clone, and 1555 from 72h-specific and 767 from 48h-specific library were obtained. All of the fragments were spotted on the microarray and hybridized with fluorescent labeled RT-probes prepared from mRNA (called RT-probe) andSSH-cDNA (called SSH-probe). The results showed that there exhibit significantly difference between the hybridization results between SSH- and RT-probe. SSH probe can hybridize with 98.8% spots on the microarray and RT-probe only 57.9%. The pattern of gene expression was very different between the two incubation times and total of 224 differentially expressed cDNA fragments were identified by RT-probe. The real-time PCR analysis displayed that some of genes were 72h-specific expression ones which are less than 48h-specific. Twelve are significantly 72h-specific and 23 are 48h-specific. All the cDNA sequences can be located on P. chrysosporium genome and some of them located on more than two scaffolds of the genome. It suggested that they may be gene families. The 72h-specific genes exhibited homolog to the genes encoding for: aryl-alcohol dehydrogenase, putative ABC transporter, heat-induced catalase, 2-Cys thioredoxin peroxidase gene and so on. Transcripts of 48h-specific genes exhibited homolog to the genes encoding for: ubiquitin-protein ligase, proline imino-peptidase, and cell wall surface anchor family protein. Some genes whose products are involved in H2O2 metabolism were found to be only in 72h culture and their function may be related to decrease the damage of H2O2 or other active oxygen species produced during secondary metabolism to P. chrysosporium.Secondly, an EST sequence analysis platform for batch the EST data was constructed on the basis of linux. Total of 757 cDNA fragments of two SSH libraries were sequenced. The batch analysis of the 757 ESTs includes vector sequence removing, EST able-blasted database construction, sequence sorting, assembling, locating on genome, exon and intron identification and gene prediction. The results demonstrated that the robust platform could accelerate data analysis for large-scale EST sequences and offer useful information for cloning the correlative genes. There are 22 genes identified as 72h-specific, 51 as 48h-specific and 65 as non-specific expression genes. Besides, there are 51 found no hybridization with RT-probe and their expression pattern is unknown. There are 25 genes display high homology with Rt-probe prepared from Trametes gallica-another strong lignin-degradation fungus.Sixty-two genes can be classified by KOG and 6 genes belong to the eukaryotic ubiquitin system. All of the EST sequences were used to predict CDS of the gene through JGI white-rot vl. The results showed that 55 predicted CDS were almost same as the known ones. The CDS of SSH362 was amplified by using the primer, whose sequences were from the predicted ones. The three-dimensional model of SSH2330 was established by employing SWISS-MODEL and has 5 6-sheet and 3 a-helix. The amino acid sequence showed that it is homologous to a subunit of yeast 20S proteasome.Thirdly, the expression of lignocellulytic biodegradation-related genes of Phanerochaete chrysosporium grown in 10 kinds of defined media for 5 days and fir wood chip throughout 2 to 8 weeks was analyzed by using the RT-PCR method. The result showed that each individual gene of lip gene family was response for special nutrient factors. The expression of lipD gene could be promoted by molecular O2 but suppressed by Mn2+. The nitorgen source was not the control factor for lipD gene expression. No clear relationship was found between nutrient factors and the expression of lip A gene. It may be regulated by several nutritional factors through a complex system. Mnp3 gene is not strongly regulated by Mn2+ and other nutrient factors and expressed in different media. Genes of CBHI family can not be expressed in the presence of glucose as the sole carbon source. Glx expression was regulated by Mn2+ and molecular O2 , and depressed when Mn2+ concentration was up to 300mg/L. The transcription patterns of genes of lip family on fir wood chip was showed to be markedly different from those in defined media. The expression of single lip gene was changed with colonized time. Only HpA2 expression could be detected on the second and 8th weeks, no transcripts on the 4th weeks. The expression of UpD2 and HpCl could be detected on the 6th weeks. No difference was observed between the expression pattern of mnp, cbh, glx genes both in defined media and fir wood chips.Finally, eleven subcloned DNA fragments of the 5'-upstream region of lipC, lip A and UpF were assayed by the DNA-protein binding and gel mobility shiftassay(GMSA). To analyze the protein binding ability of the 11 fragments, protein was extracted from P. chrysosporium mycelia grown in Kirk medium(LOP) and natural fir wood chip (TP), respectively. The results showed that the DNA fragment LG2P3(400bp), derived from the 5'-upstream regions oilipC and LG6S1-2 from lipF were able to specifically bind to LOP. The DNA fragment LG6S2(210bp) from lip? was specifically bind toTP. The results from showed that gel mobility shift assay demonstrated-that all above three DNA fragments was shifted when 2Qag or 4QMg total mycelial protein was added. On the contrary, they could not shifted when 10/ug protein was added. The free probe appeared and the shifted binds disappeared when 40 X cold probe was added. These results indicated that LG2P3, LG6S2, LG6S1-2 DNA fragments were able to specifically bind to protein produced by fungus during secondary metabolism.This research investigated systematically the gene expression of two metabolism phases and the mechanism of transcriptional regulation of lignocellulytic biodegradation-related genes of P. chrysosporium. It leads to the following novel finding: 1) Combination of SSH, cDNA microarray and bioinformatics technique is more useful to analyze the gene expression pattern of P. chrysosporium. Results showed the gene expression pattern between 72h and 48h culture was very different. 2). Occurrence of the H2O2 relevant gene suggests many 72h genes may be involved in decreasing the damage of H2O2 or other active oxygen species produced during secondary metabolism 3) The individual genes of lip gene family are response for special nutrient factors. The transcription patterns of lip gene family on fir wood chip was shown to be markedly different from those patterns in defined media. 4)Three DNA fragments have been found to be, specifically bound to the total proteins extracted from P. chrysosporium in the period lignin-degrading system formation.