拮抗木霉菌碳代谢抑制因子对ABC转运蛋白调控机理

The ATP-binding cassette (ABC) transporters use the binding and hydrolysis of ATP to power the translocation of a diverse assortment of substrates across membranes, which prevent self-intoxication and also act as strong competitors to pathogenic. Its function as either importers, bringing nutrients and other molecules into cells, or as exporters that pump toxins. Carbon catabolite repression (CCR) is a wide-domain regulatory circuit that has been shown to govern the repression of genes involved in polysaccharide degradation at transcriptional level and induce the expression of ABC transporters. In this study, Trichoderma atroviride23 was used as original strain, the regulation mechanism of Cre1 on the expression of Taabc2 in ABC transporter was explored using the combination of bioinformatics and molecular genetics techniques. Firstly, analysis of the structure characteristics and function domains for Cre1 and Taabc2 gene sequence. The effect of Cre1 on mycelial growth and morphology, ATP level and hydrolytic enzyme activity was assayed on the basis of construction of the cre1 mutation. The regulation mode of Cre1 on Taabc2 gene expression was determined. Secondly, the identification of the transcription start site of Taabc2 gene using 5'-Race method. Quantitative analysis of the expression of Taabc2 with different length promoter regions in cre1 mutant and wild type by Real-Time PCR and β-Galactosidase activity assay. The key structure domain of interaction between CRE1 and Taabc2 were confirmed in the Taabc2 promoter. Thirdly, expression and purification of CRE1 protein in E.coli, and make sure the sites of CRE1 bind Taabc2 directly by protein -DNA binding analyses (EMSA) in vitro. Finally, we aim to elucidate the interaction characteristics of Cre1 and ABC transporter in Trichoderma at molecular level. Further, we shed light on the new function of cre1 involved in the regulation of ABC pump efflux besides its inhibition of hydrolytic enzyme activity. Finally, we try to map the regulatory diagram among ABC transporter protein, hydrolytic enzymes mediated by CRE1.

木霉菌ABC转运蛋白依赖水解ATP能量推动物质运输，减少对自身毒害并提高拮抗特性。碳代谢抑制因子可全局性调控生长代谢相关基因表达，直接抑制水解酶基因的转录，诱导ABC转运蛋白表达。本研究采用深绿木霉T23菌株，结合生物信息学和分子遗传操作技术，探索木霉菌碳代谢抑制子cre1 调控ABC转运蛋白（Taabc2）表达的分子机理。首先通过cre1和Tabbc2基因结构特征分析，cre1对菌丝生长和形态、ATP及水解酶活性变化的测定，确定Cre1参与调控 Taabc2基因表达可能途径。其次，确定Taabc2基因转录起始位点，定量分析Cre1对启动子区域不同长度序列表达量的影响，确定二者相互作用的关键区域。最后克隆表达CRE1蛋白，查找 CRE1与Taabc2作用位点。阐明CRE1与 ABC转运蛋白互作的分子特征，揭示木霉菌Cre1调控新的功能，建立Cre1介导的转运蛋白和水解酶三者调控网络图。

木霉菌(Trichoderma.spp)是土壤及植物根系生态环境中广泛存在的丝状真菌，具拮抗植物病原菌，促进植物的生长。真菌碳代谢抑制具有多种调控模式和途径，主要在转录层面通过一种锌指蛋白(Cys2His2) 激活其效应蛋白活性及稳定性等过程。转录组表明：木霉菌中碳代谢抑制子CRE1全局性调控细胞生长代谢过程，保障木霉在不同生境中的存活及拮抗病原菌特性。本研究以拮抗深绿木霉T.atroviride 23为研究对象，采用生物信息学和分子遗传操作相结合策略，利用农杆菌介导的遗传转化技术（Agrobacterium tumefaciens-mediated transformation, ATMT）敲除其碳代谢抑制因子CRE1。比较突变株（∆cre1）与野生株（wide type, WT）的生长性状，初步探明cre1基因是调控深绿木霉菌菌丝生长、发育和代谢的全局性转录因子。cre1基因沉默后，∆cre1较WT菌丝生长变慢，产孢滞后，且cre1调控菌丝和产孢特性依赖于培养基组分。此外，碳代谢因子CRE1还间接调控木霉菌代谢相关基因的表达：cre1基因抑制几丁质酶、β-1,3-葡聚糖酶基因转录，调控与抗生素和毒素合成密切相关的非核糖体肽合成酶（NPRS）和聚酮合成酶基因（PKS）的表达。深入探究CRE1对参与代谢产物运输相关的ABC转运蛋白taabc2的诱导表达调控机制，系统分析Taabc2基因转录启动子区域在野生株和突变株中的表达情况，确定CRE1与Taabc2编码基因启动区的两个位点直接作用的关键区域，分别是启动子上游538和570位点，另一个位点337也是潜在的结合位点。利用原核表达系统获得CRE1蛋白，通过凝胶阻滞试验进而确定了CRE1和Taabc2启动子结合的直接证据，明确CRE1诱导Taabc2表达，间接影响某些重要代谢产物运输以及增加菌株自身的抗药性。.综上，深绿木霉T23的碳代谢因子CRE1作为一个全局性转录调控因子，抑制菌丝的生长发育，调控代谢及其产物运输相关的基因，诱导ABC转运蛋白taabc2基因的表达，在木霉菌-病原菌互作中起到关键的作用。本研究指导以CRE1为靶标，提高木霉菌细胞壁降解酶及生防相关次级代谢产物的合成和分泌，提高木霉菌生防效果具有重要意义。