| The global energy crisis accelerated the exploration for renewable resources.As the most abundant and widely distributed sustainable resource,lignocellulose exhibited tremendous potential,using lignocellulose to produce fuel ethanol has been widely generalized.However,the relatively low efficiency of cellulose hydrolysis is still one of the bottlenecks limiting its practical application.Famous for its potent ability to secrete celluase,Trichoderma reesei has been used as the model strain in cellulase expression and widely used in industry.Synthesis and secretion of cellulase is a gene-induced expression process in T.reesei.Other than cellulose,cello-oligosaccharides,cellobiose,lactose and sophorose can all induce the expression of cellulase.Over decades,researches focused on the enzymatic system composition,enzyme activity and transcriptional regulation of cellulase have been carried out and a series of achievements have been made.However,the initial stage of cellulase expression,i.e.,the perception and signal transduction process of cellulase induction,has been neglected in the research process.There is still no clear conclusion on how the inducer initiated the transcription of the cellulase genes.Therefore,the systematic study of the carbon source induction mechanism in T.reesei has important theoretical significance and practical application value.Induction of different carbon sources often involves diverse membrane receptors and intracellular signaling pathways.In recent years,many membrane proteins involved in carbon source sensing and transportation have been identified,among which the most important membrane protein for the regulation of cellulase expression is Crtl.Crtl is a membrane protein identified by transcriptome sequencing in our research,deletion of cril leads to the loss of cellulase expression capacity of T.reesei under different culture conditions.Further transcriptional analysis revealed that the transcription of main cellulase genes in ?crt1 was seriously impaired under the induction of cellulose.What's more,deletion of crt1 caused the down-regulation of xyr1,which is the most crucial transcription activation factor of the cellulase genes,indicating that Crt1 regulated the expression of cellulase on the transcriptional level in T.reesei.As a membrane protein,Crtl may be involved in the sensing steps of cellulose signals.During the study of the signal transduction pathways in T.reesei,MAPK(mitogen-activated protein kinase)pathway was found not only participated in the regulation of stress sensing,Tmk3,homolog of S.cerevisiae Hog1,was also found to play vital roles in the cellulase induction process.Deletion of tmk3 caused down-regulation of the cellulase gene at the transcriptional level,indicating the upstream pathway of Tmk3 may also be involved in signal transduction during the cellulase induction process.Due to the importance of Crtl in the induction of cellulase,it is necessary to make a comprehensive analysis of the cell localization and the signal transduction pathways involved the signal transduction of Crtl to elucidate the regulatory mechanism of cellulase gene induction.In addition,within the study of the upstream regulatory pathway of Tmk3,the carbon source induction mechanism in the induction process of T.reesei could be fully revealed.Therefore,we carried out a series of work around the upstream signal pathways of Crtl and Tmk3 in T.reesei,and mainly obtained the following research results:1.The localization of Crtl was determined,which was not only located on the cell membrane but also on the nuclear membrane during the cellulase induction process.By fusion of GFP tags to the C-terminus of Crt1 in-situ,we constructed crt1-gfp strain,and fluorescence detection was performed for Crt1 localization on different carbon sources.When cultured with the medium containing glucose and glycerol as the sole carbon source,the fluorescence signal was undetectable,the fluorescence signal of Crt1 was detected mainly located in cell membrane when the crt1-gfp strain was cultivated in the medium containing xylan as the sole carbon source.At last,during the cultivation on cellulose,lactose,cellobiose and sophorose,the fluorescence signal was detected on the cell membrane and unknown intracellular area with the"circular" distribution,as the induction process continued,the percentage of the intracellular distribution increased.Further,we investigated the subcellular localization of Crtl using immuno-gold microscopy,and the detection of colloidal gold particles by SEM indicated that the "circular" distribution area of Crtl in the cell was the nuclear membrane,besides,Crt1 was also distributed in the cell membrane and endoplasmic reticulum.Besides,the membrane fraction,nuclear fraction and outer nuclear membrane fraction of the cells were extracted from the crtl-gfp strain using the hierarchical separation technique,the results proved that Crtl existed in all the components above.In addition,Crtl was also found to be able to transfer from the cell membrane to the nuclear membrane during the carbon source transformation.2.Through co-localization analysis of Crtl and marker proteins of different cellular components,it was found that endocytosis pathway,nuclear pore complex and Sec61 complex were involved in its localization formation process,laying a foundation for the subsequent analysis of signal transduction pathway.The co-localization detection of Crt1 and transcription factor Xyrl was carried out,and the Xyr1 was found to be located inside of Crtl,which not only further confirmed the nuclear membrane localization of Crtl,but also indicated the correlation of these two proteins in localization.Subsequently,we conducted co-localization detection of Crt1 and early endosomal marker protein Rab5,and found that Crtl can co-localized with Rab5 in the middle and late stage of induction process,indicating that the endocytosis pathway may play a certain role in the localization change of Crt1.Furthermore,deletion of end3 in the endocytosis pathway indicated that inhibition of endocytosis did not change the cell localization of Crtl,but caused the deficiency of cellulase expression of Treesei.Due to the nuclear membrane localization of Crtl,co-localization analysis was performed on Crtl between Nspl and Nup205,which are two components of the nuclear pore complex.Results revealed that Nspl and Nup205 were uniformly distributed on the nuclear membrane in the non-induced state,while Nspl and Nup205 were irregularly distributed on the nuclear membrane with the expression of Crtl in the induced state,indicating that there is a competitive relationship between Crtl and different components of the nuclear pore complex on the nuclear membrane,and it also implies that the nuclear pore complex may be involved in the localization changing and signal transduction process of Crtl.Previous studies have shown that the Sec61 complex plays an important role in the nuclear transition of membrane receptors.We further conducted a study on the relationship between Sec61 complex and Crtl,results revealed that Sec61? was able to form obvious co-localization with Crt1 during the induction process,which proved that Sec61? exists not only in the endoplasmic reticulum,but also in the nuclear membrane,and may be involved in the formation of the nuclear membrane localization of Crtl.Subsequently,promoter substitution was performed for sec61?,due to the importance of the function of Sec61?,and the change of its expression quantity caused the growth defects.However,when sec61? was repressed,the cellulase formation was blocked and the fluorescence of Crtl couldn't be detected,indicating the importance of Sec61a for cellulase production and the localization formation of Crtl.Through the study of deletion of sec61?,although the growth of?sec61? and the localization of Crtl is not affected,the extracellular PNPC activity dropped by about 60%,illustrated that Sec61? was less important than Sec61?,but also played important roles in cellulase production process.Finally,proteins that may interact with Crtl have been screened through Co-IP experiments,and the function of these filtered proteins remains to be further studied.3.Through the analysis of functional area of Crtl and its own transcriptional regulation,it was found that the C-terminal and N-terminal of Crtl were necessary for its function,the regulatory relationship between Xyrl and Crtl during the cellulase induction process was investigated,and several regulatory factors involved in the transcriptional regulation of Crtl were identified.Deletion of the C-terminal of Crt1 in the wild-type strain caused the same phenotype of the ?crt1.which couldn't grow on cellulse,indicating the important role of C-terminal in the functional exertion of Crt1.By complement the ?crt1 strain with different mutants of Crt1 revealed that the N-terminal.C-terminal and the lysine in the C-terminal were essential for Crt1.We also found that the devitalized mutant caused the failure of crt1 promoter to promote the transcription of downstream genes,indicating that crt1 promoter was subject to feedback regulation of cellulase expression.The failure to achieve successful expression of Crt1 and its mutants using a heterogenous promoter suggests that Crt1 may have post-transcriptional or translational modifications.Since Xyr1 is essential for the expression of cellulase gene and the co-localization relationship between Crt1 and Xyr1,we further analyzed the relationship between Xyrl and Crt1 in the transcriptional level.Along with most cellulase genes,the transcription of crt1 could not be detected in the absence of xyr1.On the same time,when xyr1 was over-expressed on glucose condition,although the transcription of cellulase genes was up-regulated,the transcription of crt1 still could not be detected,indicating that the expression of Xyr1 was a necessary and non-sufficient condition for the expression of Crt1.Besides,over-expression of xyr1 can restored the deficiency of ACrt1 in cellulose utilizing,indicating that Xyr1 was on the downstream of Crt1 in cellulase induction.At last,we performed yeast one-hybrid screening using crt1 promoter,several proteins were screened to bind to the promoter of Crt1,among which Tr1 08357 and Rce1 may play important roles in the transcriptional regulation of Crt1.4.The upstream pathways of Hogl-type MAPK was identified and functional analyzed in T.reesei,it is determined that Shol branch and Sln1 branch play opposite roles in the regulation of cellulase expression.Two regulatory upstream pathways of Tmk3 were identified by bioinformatics analysis:the Shol branch and the Sln1 branch.For the Shol branch,deletion of Trste20 caused an obvious defect in the osmotic response,whereas repression of Trsho1 increased the resistance to osmotic,oxidative and thermo stresses.In addition,TrShol was also shown to be involved in the cell wall integrity maintenance.For the Sln1 branch,repression of Trypd1 significantly impaired the vegetative growth,cell wall integrity maintenance and stress responses.Besides,TrShol and TrSte20 of the Sho1 branch and TrYpdl of the Sln1 branch were also shown to be differentially involved in the cellulase production of T.reesei.Specifically,repression of Trshol or deletion of Trste20 significantly reduced the transcription of cellulase genes whereas overexpression of Trypd1 resulted in the reduced production of cellulases.These results indicate that the Shol branch and the Slnl branch oppositely regulate the cellulase production in T.reesei.Our present study thus implicates the osmotic response pathways in regulating the cellulase production,supporting the crosstalks of osmosensing and nutrient sensing. |
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