| Deciphering the molecular mechanism underlying the degradation of plant cell wall by filamentous fungi is imperative for degradation of lignocelluloses and the biological control of filamentous plant pathogens.In filamentous fungi,the expression of cellulase genes is repressed by simple carbon sources such as glucose and sucrose,and induced by cellulose.This process is mainly regulated at the transcription level.The key transcriptional factors xyr1 and clr-2,which are the major regulators of plant cell wall degrading enzymes are conserved across the kingdom of rot filamentous ascomycete fungi.However,it has been reported that the downstream regulons of xyr1 and clr-2varied in different species.Xyr1 is the most important transcription factor which regulates cellulases and hemicellulases in Trichoderma reesei,and its absence will directly lead to inability to synthesize any lignocellulosic enzymes in T.reesei.However,in Neurospora crassa,xlr-1,the homologous gene of xyr1,only regulates the expression of the hemicellulase genes,and it has almost no effect on the induced expression of cellulases.The expression of cellulases is mainly regulated by the transcription factor clr-2 in N.crassa.In N.crassa,the deletion of the clr-2 gene can directly lead to its inability to grow in the culture medium that uses cellulose as the sole carbon source.After constitutive expression of the clr-2 gene,N.crassa can be induced to express cellulases under repressive conditions such as glucose or sucrose.Sequence alignment analysis revealed that there is a homologous gene of clr-2 in T.reesei(gene ID: 26163,temporarily named tclr2,database:https://mycocosm.jgi.doe.gov/cgi-bin/disp Transcript?db=Trire2&id=26163&use Coords=1),and studies had shown that the overexpression of tclr2 has no effect on the expression of cellulases and hemicellulases in T.reesei.In this study,molecular genetics was used to systematically study the function of transcription factors xyr1 and clr-2 in T.reesei and N.crassa,and explore the possibility of cross-species regulation of transcription factors.The main results are as follows:1.To study the expression pattern of tclr2 in T.reesei,Tu6 strain was cultured under different carbon source conditions and the transcription level of tclr2 was tested.The results showed that the tclr2 in T.reesei is repressed by glucose and induced by cellulose,which is consistent with the expression pattern of clr-2 gene in N.crassa.T.reesei and N.crassa belong to filamentous ascomycetes,and the similarity of amino acid sequence of gene encoding protein of tclr2 in T.reesei and clr-2 in N.crassa is 61.3%.Both of clr-2and tclr2 have the structure of typical transcription factor zinc finger,and their expression is repressed by glucose and induced by cellulose.Therefore,tclr2 in T.reesei should theoretically have the same or similar function as clr-2 in N.crassa,that is,tclr2 can regulate the expression of cellulases in T.reesei.To test this hypothesis,the strain Tr?tclr2 which tclr2 gene was deleted was constructed in this paper,and its cellulase gene expression was detected under the carbon source of cellulose.The results showed that the deletion of tclr2 in T.reesei had no significant effect on the expression of cellulases.Furthermore,the transcription levels of the major cellulase genes cbh1 and cbh2 in T.reesei were detected,and it was found that the transcription levels of the cbh1 and cbh2 genes in Tr?tclr2 strain did not change significantly compared with the starting strain.These results indicate that although tclr2 of T.reesei and clr-2 of N.crassa are very similar in sequence and expression pattern,their functions are completely different.2.From the above results,it was known that tclr2 of T.reesei cannot regulate the expression of cellulase gene in T.reesei.The reason may be that:(1)TCLR2 protein lost the activity of transcription factor during evolution;(2)The promoter sequences of the cellulase genes in T.reesei lost the binding motifs of TCLR2 protein during the evolution,and cannot bind to TCLR protein.To clarify the reason why tclr2 cannot regulate cellulase gene in T.reesei,tclr2 of T.reesei was constitutively expressed in N.crassa mutant which clr-2 was deleted,to detect whether the heterologous expression of tclr2 in N.crassa can complement the deletion phenotype of clr-2 gene.The results showed that the recombinant strain can hardly secrete any protein under the condition of sucrose as the sole carbon source,and the cellulase genes cbh1 and cbh2 are also not transcribed under this condition,and the recombinant strain cannot grow under the condition of cellulose as the sole carbon source,which indicates that the tclr2 gene of T.reesei did not complement the gene function of clr-2 of N.crassa in the recombinant strain,and the tclr2 gene may lose the function of transcription factor during evolution.3.To further investigate whether the functional CLR2 protein of N.crassa can regulate the expression cellulase gene in T.reesei,the tclr2 gene was replaced by the constitutively expressed functional clr-2 gene of N.crassa in T.reesei.And under the condition of glucose as carbon source,it was tested whether clr-2 could mediate the expression of cellulase genes under repressive conditions as in N.crassa.At the same time,the clr-2 gene of N.crassa was heterologously expressed in xyr1-deficient strain named Tr?xyr1,and it was tested whether clr-2 could complement the gene function of xyr1 under cellulose.The experimental results showed that the heterologous expression of clr-2 gene from N.crassa did not result in the expression of cellulase genes in T.reesei,which indicates that the clr-2 gene of N.crassa cannot achieve cross-species regulation of the cellulase gene in T.reesei.The reason may be that the promoters of cellulase genes in T.reesei lost their binding motifs.To test this hypothesis,the promoter of the cellobiohydrolase gene cbh1 from T.reesei was further replaced with cbh1 promoter from N.crassa in the T.reesei which heterologously constitutively expressing the clr-2 gene from N.crassa,and it was detected whether the single expression of CBH1 cellulase can be achieved under glucose.The experimental results showed that the newly constructed recombinant strain could secrete weak CBH1 protein under glucose,which proves that the clr-2 gene of N.crassa can interact with cbh1 promoter of N.crassa in T.reesei to regulate downstream gene expression.4.To investigate whether the xyr1 gene of T.reesei can across species to regulate the expression of the lignocellulose-degrading enzyme genes of N.crassa,xyr1 was constitutively expressed in clr-2-deficient N.crassa strain named Nc?clr2 to test whether xyr1 can complement the function of the clr-2 gene.The experimental results showed that under the condition of sucrose as the sole carbon source,the recombinant strain can hardly secrete any protein,and when cellulose was the sole carbon source,the recombinant strain cannot grow,which indicates that similar to clr-2,xyr1 gene could not achieve cross-species regulation between the two species either.In summary,although N.crassa and T.reesei are very close in evolution and can degrade lignocellulose in natural habitats,the expression regulation of lignocellulose degrading enzyme genes of them is very different.Although the key transcriptional regulators clr-2 and xyr1 exist in both of them,and their sequences and expression patterns have many similarities,their functions have changed a lot,and they cannot achieve cross-species regulation in these two species. |
PDF Full Text Request