偏肿革裥菌细胞色素P450基因在刚果红脱色条件下的差异表达
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摘要
为了明确白腐真菌偏肿革裥菌(Lenzites gibbosa)降解双偶氮染料刚果红过程中细胞色素P450基因的功能,采用高通量测序技术对刚果红染料处理0、3、7、10 h下的菌丝样本进行转录组测序,以比对到参考基因组上的每个基因的reads数即Count值作为表达量值,根据基因在对照组(0 h)和染料处理组(3、7、10 h)中表达量的变化,用DESeq_EBSeq软件进行样品组间的差异基因表达分析和差异基因筛选,对差异表达基因(DGEs)在eggNOG、GO和KEGG等数据库中进行功能注释与通路富集分析,对筛选出的相关差异基因在NCBI上进行DNA序列比对,并与相关参考文献进行比较分析。结果表明:在转录组所有的DGEs中,搜索出11个可能影响L. gibbosa降解刚果红过程的细胞色素P450基因,其中在CK vs GGH1(0 vs 3 h)中未筛选出细胞色素P450差异基因,在CK vs GGH2(0 vs 7 h)中筛选出3个差异基因,在CK vs GGH3(0 vs 10 h)中筛选出8个差异基因,表明在L. gibbosa对刚果红染料脱色降解过程中细胞色素P450基因逐步参与了降解脱色过程。在10 h时,被注释到木质素降解关键通路ko 01220(芳香族化合物降解)中的gene_25488表现出差异表达。从11个细胞色素P450基因的差异表达中可以探究并揭示出一些潜在的生物学规律,最终结果表明L. gibbosa的细胞色素P450基因与双偶氮类染料刚果红的降解脱色有一定的关系。
In order to clarify the function of cytochrome P450 genes from white rot fungus Lenzites gibbosa growing under the degradation of diazo dye Congo red,the transcriptomes were constructed on mycelia samples treated with Congo red at 0,3,7,and 10 h using high-throughput sequencing technology. The number of reads of each gene aligned to the reference genome,i.e. Count value,was used as the value of expression quantity,DESeq_EBSeq software was used to analyze the expression of differential genes and screen the differentially expressed genes( DEGs) according to the changes of gene expression quantity between the control group( 0 h) and the dye treatment group( 3,7,and 10 h). The DEGS were functionally annotated in eggN OG,GO,KEGG,and other databases,and significantly enriched pathways of DEGs were analyzed. The screened DEGs were alligned on NCBI for DNA sequences and compared with relevant references. The 11 cytochrome P450 genes that may affect the degradation process of Congo red by L. gibbosa were searched out from all the DGEs of transcriptomes,among them,no differentially expressed cytochrome P450 genes were screened in CK vs GGH1( 0 vs 3 h),3 cytochrome P450 genes were screened in CK vs GGH2( 0 vs 7 h),and 8 cytochrome P450 genes were screened in CK vs GGH3( 0 vs 10 h),indicating that cytochrome P450 genes are gradually involved in the degradation and decolorization process of Congo red by L. gibbosa. At 10 h,gene_25488 annotated into the key pathway of lignin degradation ko01220( degradation of aromatic compounds) showed differential expression. Some potential biological regulations can be explored and revealed from the differential expression of 11 cytochrome P450 genes. The final results showed that the cytochrome P450 gene of L. gibbosa have some relationship with the degradation and decolorization of the diazo dye Congo red.
In order to clarify the function of cytochrome P450 genes from white rot fungus Lenzites gibbosa growing under the degradation of diazo dye Congo red,the transcriptomes were constructed on mycelia samples treated with Congo red at 0,3,7,and 10 h using high-throughput sequencing technology. The number of reads of each gene aligned to the reference genome,i.e. Count value,was used as the value of expression quantity,DESeq_EBSeq software was used to analyze the expression of differential genes and screen the differentially expressed genes( DEGs) according to the changes of gene expression quantity between the control group( 0 h) and the dye treatment group( 3,7,and 10 h). The DEGS were functionally annotated in eggN OG,GO,KEGG,and other databases,and significantly enriched pathways of DEGs were analyzed. The screened DEGs were alligned on NCBI for DNA sequences and compared with relevant references. The 11 cytochrome P450 genes that may affect the degradation process of Congo red by L. gibbosa were searched out from all the DGEs of transcriptomes,among them,no differentially expressed cytochrome P450 genes were screened in CK vs GGH1( 0 vs 3 h),3 cytochrome P450 genes were screened in CK vs GGH2( 0 vs 7 h),and 8 cytochrome P450 genes were screened in CK vs GGH3( 0 vs 10 h),indicating that cytochrome P450 genes are gradually involved in the degradation and decolorization process of Congo red by L. gibbosa. At 10 h,gene_25488 annotated into the key pathway of lignin degradation ko01220( degradation of aromatic compounds) showed differential expression. Some potential biological regulations can be explored and revealed from the differential expression of 11 cytochrome P450 genes. The final results showed that the cytochrome P450 gene of L. gibbosa have some relationship with the degradation and decolorization of the diazo dye Congo red.
引文
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[2]谭国民,柳荣展,李群,等.白腐菌对染料废水脱色及降解的研究[J].中国造纸学报,2003,18(1):127-130.
[3]张玉龙.偏肿革裥菌MnP发酵条件的优化和纯化以及对染料脱色的研究[D].哈尔滨:东北林业大学,2012.
[4]闫洪波.偏肿拟栓菌锰过氧化物酶c DNA基因克隆及在毕赤酵母中的表达[D].哈尔滨:东北林业大学,2009.
[5]肖鹏飞,近藤隆一郎.细胞色素P450抑制剂对白腐菌Phlebia lindtneri降解氯丹的影响[J].农药学学报,2012,14(5):515-520.
[6]贺丽虹,赵淑娟,胡之璧.植物细胞色素P450基因与功能研究进展[J].药物生物技术,2008,15(2):142-147.
[7]KIM D,PERTEA G,TRAPNELL C,et al.TopHat2:accurate alignment of transcriptomes in the presence of insertions,deletions and gene fusions[J].Genome Biology,2013,14:R36.doi:10.1186/gb-2013-14-4-r36.
[8]LANGMEAD B,TRAPNELL C.Ultrafast and memory-efficient alignment of short DNA sequences to the human genome[J].Genome Biology,2009,10(3):R25.doi:10.1186/gb-2009-10-3-r25.
[9]ALTSCHUL S F,MADDEN T L,SCHFFER A A,et al.Gapped BLAST and PSI BLAST:A new generation of protein database search programs[J].Nucleic Acids Research,1997,25(17):3389-3402.
[10]TATUSOV R L,GALPERIN M Y,NATALE D A.The COG database:a tool for genome scale analysis of protein functions and evolution[J].Nucleic Acids Research,2000,28(1):33-36.
[11]ASHBURNER M,BALL C A,BLAKE J A,et al.Gene ontology:tool for the unification of biology[J].Nature Genetics,2000,25(1):25-29.
[12]KANEHISA M,GOTO S,KAWASHIMA S,et al.The KEGGresource for deciphering the genome[J].Nucleic Acids Research,2004,32:D277-D280.
[13]邓泱泱,荔建琦,吴松锋,等.nr数据库分析及其本地化[J].计算机工程,2006,32(5):71-74.
[14]APWEILER R,BAIROCH A,WU C H,et al.UniProt:the universal protein knowledgebase[J].Nucleic Acids Research,2004,32:D115-D119.
[15]宁大亮.白腐真菌细胞色素P450转化难降解有机物的功能研究[D].北京:清华大学,2009.