| Trichoderma sp., a ubiquitously distributed soil inhabitant, has been widely usedin the control of plant disease, and soil pollution prevention and remediation.However, for a better cooperative application with cupric pesticides in plant diseasecontrol or increasing product with fertilizer, and bioremediation of heavy metalpollution, we need construct strains of Trichoderma which showed high capability ofcopper tolerance and accumulation. Therefore, study on the copper tolerance,biosorption and transport mechanism may establish the theoretical foundation ofdisease biocontrol and bioremediation. It is also significance for constructing highcopper tolerance and bioaccumulation genetically engineered fungi strains. Thisresearch was undertaken to establish a simple and highly efficient ATMT system forefficient screen of mutants of Trichoderma reesei, which showed high ability ofcopper accumulation. Furthermore, by cloning and analysis of the correspondinggenes, we can elaborate the copper metabolic mechanism on a genetic, biochemistryand genomics levels. The main researches are as follows:1. Construction and screening of ATMT transformants with high ability ofcopper bioaccumulationA modified Agrobacterium tumefaciens-mediated transformation method wasestablished for the construction of mutants with improved copper tolerance andaccumulation capability in Trichoderma reesei. One transformant AT01was isolatedfrom1664transformants, which showed the highest copper accumulation capability.Incubated on the medium contained0.7mM copper, AT01display the maximumcopper removal capability with a12.73mg copper/g biomass (removal rate of96.1%),whereas the wild-type strain had merely5.97mg copper/g biomass (removal rate of49.6%). The optimal initial pH was5.0, and the optimal incubation temperature was 28°C. Electron microscope observation revealed that copper was mainly accumulatedin cell vacuoles, consequently improve copper tolerance ability of strain AT01.2. Tad1cloning and function analysisWith inverse PCR, we cloned a novel gene Tad1. It contains1533bp openreading frames (belongs to COG0402) encoding a protein of510amino acids whichbelongs to the amidohydrolase superfamily. Protein was purified through prokaryoticexpression and its activities were also determined. The data indicates that adenine isthe best substrate with a Km0.66mM. By gene overexpression and RNAi-mediatedsilence experiments, we found that gene was closely related to copper accumulation inTrichoderma because accumulation level of cooper by WT, AT01, Ovex-Tad1andRNAi-Tad1were7.5,14.1,12.3and4.9mg/g, respectively (P=0.02, t-test). To furtherconfirm correlation of adenine deaminase with copper transport-related genes, weinvestigated the expression levels of Tratx, Trccs, Trcox, and Trace (homologs of thecopper chaperone in yeast) under extracellular copper stress in T. reesei. Resultsshowed that the expressions of those copper chaperones were increased under copperstress both in Ovex-Tad1and AT01. However, there were no difference between in theRNAi-Tad1and WT. Furthermore, RP-HPLC revealed concentration of hypoxanthineand xanthine were significantly increased in AT01and Ovex-Tad1, and decreased inRNAi-Tad1, which thus demonstrated that Tad1was participated in the copperaccumulation.3. Tad1regulated the copper metabolism related genesThe gene expression profile of AT01was compared with a wild-type T. reeseistrain in medium containing1.0mM Cu2+. A total of624genes were up-regulated and305genes were down-regulated under copper stress. All all differential genes could bedivided into22categories, in which up-regulated genes were grouped to threecategories.(1) Metal ions homeostasis related genes: It includes26transmembranetransporters and intracellular trafficking related genes (4.2%),25inorganic ions transport and metabolism (4.0%).(2) Copper tolerance and detoxification relatedgenes: It includes11detoxification related genes (1.8%),8copper tolerance relatedgenes (1.3%) and eight genes responsible for cell wall, cell membrane, envelopebiogenesis (1.3%). When the latter eight genes were deleted, the knockout mutantgrowth changed slower than WT and the copper accumulation ability was alsodecreased. However the mechanisms need further research.(3) Adenine metabolismrelated genes: It contains nine genes (1.4%), in which there were eight genes showingsignificant difference at RNA expression level.①Ferroxidase-like protein (Fet3p),it catalyzes the oxidation of Fe2+into Fe3+, the posttranslational insertion of fourcopper molecules into Fet3p is essential for its activity. Extracellular copperconcentration has a significant influence on the gene expression.②Glutathionetransferase is a wide distribution enzyme. It plays a significant role in detoxification,which is a vital metabolic and defense capability of living organisms. The keyreaction catalyzed by Gsts is the conjugation of the tripeptide glutathione.③C-typecytochrome is a widespread class of essential metalloproteins located on the positiveside of energy-transducing membrane systems. It catalyzes the reduction of molecularoxygen and couples this reduction with proton translocation across the IM. They arealso involved in cell death pathways in vertebrates by signaling mitochondrial status.④Encodes a protein homologous to a metallochaperone Ccs1of S.pombe. Ccs1, a27kDa three-domain polypeptide, is a copper metallochaperone for Cu, Zn superoxidedismutase (SOD), which is participated in the copper detoxification.⑤Tctr2, codingfor a orthology of Ctr2of S.pombe. It may participate in the copper stored in vacuoles.⑥Allantoinase, catalyzes the conversation of allantoin to allantoic acid, was a keyenzyme participated in the purine catabolism.⑦Phosphoribosylformylglycinamidinesynthase (PAICS), participates in the conversion of PRPP into inosine5′-monophosphate (IMP) synthesis in de novo synthesis of purine.⑧GTPcyclohydrolase, catalyzes the metabolism of GTP, the product of xanthosinemonophosphate (XMP). RNA Expression levels of all above eight genes were twotimes higher than that of WT. 4. Clone of the copper transport related genesIt was the first time to clone six copper metabolism related genes: Tmac1, Trace,Tctr3, Trccs, Tratx and Trcox. Tmac1, a homologous protein of Mac1, was atranscriptional activator. It sensed very low concentration of copper and regulated thehigh affinity copper transport of Saccharomyces cerevisiae. Trace, a homologousprotein of Ace1of S.cerevisiae, which control the MTs expression. Tctr3, ahomologous protein of Ctr3of S.cerevisiae, which is transmembrane proteinparticipating in copper transport. Trccs, a homologous protein of Ccs1of S.cerevisiae,deliver the copper ions to SOD specifically. Tratx, a homologous protein of of Atx1ofS.cerevisiae, deliver the copper ions to Golgi body specifically. Trcox, a homologousprotein of Ccs1of S.cerevisiae, deliver the copper ions to cytochrome c oxidasespecifically. Tmac1coding for a protein contains501amino acids and regulated thetransmembrane protein Tctr3. Furthermore, two Cys-His repeats metal binding motifsof protein Tmac1, one in the354to369C terminus and one in the475to490Cterminus were also present in Mac1. A deletion mutant of Tmac1was hypersensitiveto the copper starvation and showed poor growth. Subsequently, the function wasrecovered by the gene complementation experiment. Furthermore, the Tmac1genefully complemented growth defects of yeast Mac1mutant. Trace, coding for a405amino acids protein, contains a DNA binding domain CVRGHR in N terminal andfour CXXC domains which associate with copper binding. The Trace gene fullycomplemented growth defects of yeast CUP1mutant. Tctr3, coding for a178aminoacids protein, contains MLLAM domain in N terminal. This protein contains threetransmembrane domains and participates in the copper transmembrane transportation.Trccs、Tratx and Trcox coding for three small inducible proteins copper molecularchaperones, respectively. Trccs, coding for a248amino acids protein, contains acopper binding domain CXXCV in N terminal. Tratx, coding for an82amino acidsprotein, contains a copper binding domain MTCXXC in N terminal. Trcox, coding fora61amino acids protein, contains a copper binding domain CXXCV in N terminal.To sum up, we propose that Tad1gene how to regulate the copper accumulation. Overexpression of Tad1(Adenine deaminase) induced the accumulation ofhypoxanthine and xanthine. Metal ions like copper can bind to both of them togenerated mixed-ligand metal complexes. Excess of hypoxanthine and xanthine willbind large amount of copper ions which reduce the intracellular free copper. Signal ofcopper deficiency would then active the TFs (transcriptional factors), which openedthe transmembrane copper transporters. And then, lots of copper was uptake. As thecopper concentration increase, a series of copper depend proteins was active toalleviate concentration of the intracellular copper ions to protect the cells. Forinstance, transcriptional factor Trace was activated and then regulated the MTsexpression, which can protect cell against the copper poison. Furthermore, expressionlevels of Trccs、Tratx、Trcox also increased, which reduce the concentration of copperions by delivering it to different target organelles. |
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