里氏木霉铜代谢相关基因克隆与功能分析
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摘要
木霉菌是土壤中普遍存在的习居菌，在植物病害生物防治和土壤农化污染修复中具有广泛的应用。然而木霉菌在与铜制剂农药协同防治植物病害、与肥料复合使用提高作物产量及修复重金属污染环境中，需要具备良好的铜耐受性或吸附性。因此，研究木霉铜代谢相关基因及其功能，揭示木霉菌耐受、吸附和转运铜的机理，为提高木霉菌在铜胁迫下的防病、修复环境效应奠定理论基础，对构建高效吸附或高耐受铜的工程菌也具有重要意义。本研究以基因组已测序的里氏木霉（Trichoderma reesei）QM6a为原始菌，克隆铜代谢相关基因，并通过改进的农杆菌介导转化技术（ATMT）构建、筛选高吸附铜的里氏木霉突变株，克隆高吸附铜相关基因，在遗传、生物化学和组学的尺度上明确其代谢功能和作用机制。主要的研究内容如下：
1.高吸附铜突变株的构建与筛选
对常规的农杆菌介导（ATMT）转化方法进行了改进，建立了一种高效筛选吸附铜突变株的方法。通过该方法，构建了1664株突变株，并从中筛选到一株高吸附铜突变株AT01，当培养基铜浓度为0.7mM时，AT01铜吸附率达到12.73mg/g（96.1%），而野生株（WT）吸附率仅为5.97mg/g（49.6%）。显微镜观察表明，AT01突变株胞内液泡数量明显多于WT，在铜胁迫下可富集较多的铜离子，胞内多余的铜离子储存在液泡中可减少铜对细胞的毒性作用，进而提高木霉菌对铜的耐受能力。
2. Tad1基因克隆与功能分析
通过反向PCR扩增获得的T-DNA插入片段侧翼序列并通过NCBI比对分析后获得基因Tad1。分析表明，该基因读码框含1533bp碱基，无内含子，在里氏木霉中为单拷贝，编码一个510氨基酸的蛋白，属于COG0420，为酰胺水解酶超家族成员。原核表达产物纯化后，进行酶活测定，结果表明：以腺嘌呤为底物时，酶活性最高，Km为0.66mM。RNA介导基因沉默和过表达分析表明：在1mM铜胁迫下，WT、AT01、过表达子(Ovex-Tad1)及沉默子（RNAi-Tad1）的铜吸附水平分别为7.5、14.1、12.3及4.9mg/g （P值为0.02，T-test）。在同浓度铜胁迫下，通过荧光定量PCR分析了RNAi-Tad1、Ovex-Tad1及AT01、WT中已知的酵母菌铜代谢相关基因的表达水平，结果表明：Trace、Trccs、Tratx、Trcox四个基因在Ovex-Tad1和AT01中均上调表达，而在RNAi-Tad1中，这些基因表达水平与野生株无明显差异。进一步通过HPLC分析WT、AT01、Ovex-Tad1、RNAi-Tad1中可与铜相结合的次黄嘌呤、黄嘌呤产生水平，发现与WT相比，Ovex-Tad1和AT01中两种次生代谢产物的产生均明显增加，而RNAi-Tad1产量低于野生株。上述研究从遗传和生化水平上说明该基因参与里氏木霉对铜的吸附过程。
3. Tad1基因对铜代谢相关基因的调控作用
通过表达谱芯片技术分析了AT01与WT在1mM铜胁迫下基因表达水平的差异，共获得624个上调基因，305个下调基因。根据NCBI对基因功能的分析，上调基因共分为22类。其中与铜代谢相关的基因主要有三类：（1）调控胞内离子平衡的基因。如胞内转运，细胞器跨膜运输相关基因26个（4.2%），无机离子运输及代谢相关基因25个（4.0%）。（2）耐受和解除重金属毒性作用的相关基因，如细胞解除重金属毒性相关基因有11个（1.8%）；与细胞耐受胁迫反应相关基因有11个（1.8%）；与细胞壁合成，细胞器的膜合成相关基因有8个（1.3%），这些基因可能参与铜的跨膜运送。对这8个基因进行了基因敲除，结果表明，其中一个基因被敲除后，菌体在铜胁迫下生长受到抑制，而且铜的吸附能力也低于WT，该基因功能有待进一步研究。（3）腺嘌呤脱氨酶功能及腺嘌呤代谢相关基因9个（1.4%）。为验证上述基因的表达水平，选择了上述铜代谢及腺嘌呤代谢相关基因中差异最显著的8个基因进行RT-PCR分析：①铁氧化酶蛋白（Fet3p），催化二个铁氧化成三价铁，1分子该酶需要4分子铜作为辅基。酵母菌研究表明，胞外铜离子浓度变化对该酶活性有明显影响。②谷胱甘肽转移酶，广泛分布于生物体内具多种功能的超家族酶，是真菌胞内非常重要的铜解毒肽类物质。主要催化谷胱甘肽（GSH）的结合反应。③细胞色素C氧化酶，存在线粒体内膜，是细胞呼吸链中非常关键的一个酶，在动物细胞凋亡过程起重要作用。该酶催化功能需要铜离子作为辅基。④Cccs同源蛋白。Cccs蛋白是酵母细胞内小分子多肽，主要功能是负责将胞内铜离子运送到SOD酶（超氧化物歧化酶）。与胞内铜解毒功能密切相关。⑤Tctr2基因，编码一个Ctr2同源基因，该基因在酿酒酵母中与亚铜离子液泡储存相关。⑥尿囊素酶基因，该基因催化尿囊素转变成尿囊酸，为腺嘌呤分解代谢下游关键酶。⑦磷酸核糖甲酰甘氨脒合酶属于腺嘌呤代谢途径中分支途径中一个酶，催化磷酸核糖焦磷酸（PRPP）转变为5`单磷酸盐肌苷（IMP）。⑧GTP（三磷酸鸟苷）环水解酶，催化GTP水解为单磷酸黄苷，腺嘌呤代谢途径中分支途径中一个酶。结果表明,与野生株相比，这些基因在过表达子和AT01中表达水平都比野生株提高2倍以上。
4.胞内铜转运相关基因克隆及功能分析
首次在里氏木霉菌中克隆并验证6个铜代谢调控因子及功能基因：Tmac1、Trace、Tctr3、Trccs、Tratx、Trcox。其中Tmac1与酿酒酵母(Saccharomyces cerevisiae)Mac1蛋白同源，胞内高亲和铜转运调控因子；Trace与酵母Ace1蛋白同源，控制胞内金属蛋白的表达，参与胞内铜毒性的解毒；Tctr3与酵母跨膜蛋白Ctr3同源，参与铜的跨膜转运；Trccs与酵母Ccs1蛋白同源，特异性将铜转运到胞质中的抗氧化酶SOD；Tratx与酵母Atx1蛋白同源，能够与亚铜离子结合形成二聚体，将其传递给高尔基体；Trcox与酵母Cox17蛋白同源，通过两个中间蛋白因子介导，将亚铜离子传递给细胞色素C氧化酶。进一步研究表明Tmac1基因编码一个501氨基酸的蛋白。在蛋白C端具两个Cys-His重复序列结构，与铜结合有关。敲除该基因后，木霉菌生长缓慢且对铜饥饿敏感，基因回补后，功能得到完全恢复。将该基因回补到酵母突变株△Mac1能完全恢复酵母突变株耐受铜饥饿能力。Trace基因编码一个含405氨基酸的蛋白，蛋白含4个CXXC结构域，与铜结合有关。同时在蛋白N端含铜依赖型DNA结合结构域CVRGHR。酵母回补实验表明，该基因能够完全恢复酵母△Ace1的功能。Tctr3蛋白含178个氨基酸，具有3个跨膜结构域，N端含保守结构域MLLAM。Trccs、Tratx、Trcox分别编码三个小分子铜分子伴侣。Trccs编码248个氨基酸的蛋白，N端含保守结构域CXXCV，与铜结合功能相关酵母胞内铜相关分子伴侣同源。Tratx编码一条含82氨基酸的蛋白，N端具有保守结构域MTCXXC。Trcox编码61个氨基酸的蛋白。另外，Trace、Trccs、Tratx、Trcox这四个基因的表达水平变化可用于监测细胞内铜离子浓度的变化。
综合上述实验结果提出Tad1基因参与木霉菌铜吸附和胞内铜代谢可能途径为：腺嘌呤脱氨酶（Tad1基因编码）催化木霉胞内腺嘌呤代谢途径中的次黄嘌呤及黄嘌呤合成，而这两种物质与胞内铜离子结合，导致细胞铜离子浓度低于正常水平。胞内自由铜离子浓度的微小改变激活了细胞内稳态调控网络，引起细胞泵入铜离子，而导致胞内铜离子浓度升高。铜浓度的升高，激活胞内转录因子Trace，调控胞内金属硫蛋白表达；同时，胞内铜相关分子伴侣Trccs、Tratx、Trcox表达上调，负责将胞内多余的铜离子转运到不同细胞器，解除胞内过量铜引起的细胞毒性。
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 bioaccumulation
A 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 analysis
With 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 genes
The 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 genes
It 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.
1.高吸附铜突变株的构建与筛选
对常规的农杆菌介导（ATMT）转化方法进行了改进，建立了一种高效筛选吸附铜突变株的方法。通过该方法，构建了1664株突变株，并从中筛选到一株高吸附铜突变株AT01，当培养基铜浓度为0.7mM时，AT01铜吸附率达到12.73mg/g（96.1%），而野生株（WT）吸附率仅为5.97mg/g（49.6%）。显微镜观察表明，AT01突变株胞内液泡数量明显多于WT，在铜胁迫下可富集较多的铜离子，胞内多余的铜离子储存在液泡中可减少铜对细胞的毒性作用，进而提高木霉菌对铜的耐受能力。
2. Tad1基因克隆与功能分析
通过反向PCR扩增获得的T-DNA插入片段侧翼序列并通过NCBI比对分析后获得基因Tad1。分析表明，该基因读码框含1533bp碱基，无内含子，在里氏木霉中为单拷贝，编码一个510氨基酸的蛋白，属于COG0420，为酰胺水解酶超家族成员。原核表达产物纯化后，进行酶活测定，结果表明：以腺嘌呤为底物时，酶活性最高，Km为0.66mM。RNA介导基因沉默和过表达分析表明：在1mM铜胁迫下，WT、AT01、过表达子(Ovex-Tad1)及沉默子（RNAi-Tad1）的铜吸附水平分别为7.5、14.1、12.3及4.9mg/g （P值为0.02，T-test）。在同浓度铜胁迫下，通过荧光定量PCR分析了RNAi-Tad1、Ovex-Tad1及AT01、WT中已知的酵母菌铜代谢相关基因的表达水平，结果表明：Trace、Trccs、Tratx、Trcox四个基因在Ovex-Tad1和AT01中均上调表达，而在RNAi-Tad1中，这些基因表达水平与野生株无明显差异。进一步通过HPLC分析WT、AT01、Ovex-Tad1、RNAi-Tad1中可与铜相结合的次黄嘌呤、黄嘌呤产生水平，发现与WT相比，Ovex-Tad1和AT01中两种次生代谢产物的产生均明显增加，而RNAi-Tad1产量低于野生株。上述研究从遗传和生化水平上说明该基因参与里氏木霉对铜的吸附过程。
3. Tad1基因对铜代谢相关基因的调控作用
通过表达谱芯片技术分析了AT01与WT在1mM铜胁迫下基因表达水平的差异，共获得624个上调基因，305个下调基因。根据NCBI对基因功能的分析，上调基因共分为22类。其中与铜代谢相关的基因主要有三类：（1）调控胞内离子平衡的基因。如胞内转运，细胞器跨膜运输相关基因26个（4.2%），无机离子运输及代谢相关基因25个（4.0%）。（2）耐受和解除重金属毒性作用的相关基因，如细胞解除重金属毒性相关基因有11个（1.8%）；与细胞耐受胁迫反应相关基因有11个（1.8%）；与细胞壁合成，细胞器的膜合成相关基因有8个（1.3%），这些基因可能参与铜的跨膜运送。对这8个基因进行了基因敲除，结果表明，其中一个基因被敲除后，菌体在铜胁迫下生长受到抑制，而且铜的吸附能力也低于WT，该基因功能有待进一步研究。（3）腺嘌呤脱氨酶功能及腺嘌呤代谢相关基因9个（1.4%）。为验证上述基因的表达水平，选择了上述铜代谢及腺嘌呤代谢相关基因中差异最显著的8个基因进行RT-PCR分析：①铁氧化酶蛋白（Fet3p），催化二个铁氧化成三价铁，1分子该酶需要4分子铜作为辅基。酵母菌研究表明，胞外铜离子浓度变化对该酶活性有明显影响。②谷胱甘肽转移酶，广泛分布于生物体内具多种功能的超家族酶，是真菌胞内非常重要的铜解毒肽类物质。主要催化谷胱甘肽（GSH）的结合反应。③细胞色素C氧化酶，存在线粒体内膜，是细胞呼吸链中非常关键的一个酶，在动物细胞凋亡过程起重要作用。该酶催化功能需要铜离子作为辅基。④Cccs同源蛋白。Cccs蛋白是酵母细胞内小分子多肽，主要功能是负责将胞内铜离子运送到SOD酶（超氧化物歧化酶）。与胞内铜解毒功能密切相关。⑤Tctr2基因，编码一个Ctr2同源基因，该基因在酿酒酵母中与亚铜离子液泡储存相关。⑥尿囊素酶基因，该基因催化尿囊素转变成尿囊酸，为腺嘌呤分解代谢下游关键酶。⑦磷酸核糖甲酰甘氨脒合酶属于腺嘌呤代谢途径中分支途径中一个酶，催化磷酸核糖焦磷酸（PRPP）转变为5`单磷酸盐肌苷（IMP）。⑧GTP（三磷酸鸟苷）环水解酶，催化GTP水解为单磷酸黄苷，腺嘌呤代谢途径中分支途径中一个酶。结果表明,与野生株相比，这些基因在过表达子和AT01中表达水平都比野生株提高2倍以上。
4.胞内铜转运相关基因克隆及功能分析
首次在里氏木霉菌中克隆并验证6个铜代谢调控因子及功能基因：Tmac1、Trace、Tctr3、Trccs、Tratx、Trcox。其中Tmac1与酿酒酵母(Saccharomyces cerevisiae)Mac1蛋白同源，胞内高亲和铜转运调控因子；Trace与酵母Ace1蛋白同源，控制胞内金属蛋白的表达，参与胞内铜毒性的解毒；Tctr3与酵母跨膜蛋白Ctr3同源，参与铜的跨膜转运；Trccs与酵母Ccs1蛋白同源，特异性将铜转运到胞质中的抗氧化酶SOD；Tratx与酵母Atx1蛋白同源，能够与亚铜离子结合形成二聚体，将其传递给高尔基体；Trcox与酵母Cox17蛋白同源，通过两个中间蛋白因子介导，将亚铜离子传递给细胞色素C氧化酶。进一步研究表明Tmac1基因编码一个501氨基酸的蛋白。在蛋白C端具两个Cys-His重复序列结构，与铜结合有关。敲除该基因后，木霉菌生长缓慢且对铜饥饿敏感，基因回补后，功能得到完全恢复。将该基因回补到酵母突变株△Mac1能完全恢复酵母突变株耐受铜饥饿能力。Trace基因编码一个含405氨基酸的蛋白，蛋白含4个CXXC结构域，与铜结合有关。同时在蛋白N端含铜依赖型DNA结合结构域CVRGHR。酵母回补实验表明，该基因能够完全恢复酵母△Ace1的功能。Tctr3蛋白含178个氨基酸，具有3个跨膜结构域，N端含保守结构域MLLAM。Trccs、Tratx、Trcox分别编码三个小分子铜分子伴侣。Trccs编码248个氨基酸的蛋白，N端含保守结构域CXXCV，与铜结合功能相关酵母胞内铜相关分子伴侣同源。Tratx编码一条含82氨基酸的蛋白，N端具有保守结构域MTCXXC。Trcox编码61个氨基酸的蛋白。另外，Trace、Trccs、Tratx、Trcox这四个基因的表达水平变化可用于监测细胞内铜离子浓度的变化。
综合上述实验结果提出Tad1基因参与木霉菌铜吸附和胞内铜代谢可能途径为：腺嘌呤脱氨酶（Tad1基因编码）催化木霉胞内腺嘌呤代谢途径中的次黄嘌呤及黄嘌呤合成，而这两种物质与胞内铜离子结合，导致细胞铜离子浓度低于正常水平。胞内自由铜离子浓度的微小改变激活了细胞内稳态调控网络，引起细胞泵入铜离子，而导致胞内铜离子浓度升高。铜浓度的升高，激活胞内转录因子Trace，调控胞内金属硫蛋白表达；同时，胞内铜相关分子伴侣Trccs、Tratx、Trcox表达上调，负责将胞内多余的铜离子转运到不同细胞器，解除胞内过量铜引起的细胞毒性。
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 bioaccumulation
A 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 analysis
With 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 genes
The 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 genes
It 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.
引文
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