ClpX ATPase在蜡样芽孢杆菌生物防治小麦土传病害中的作用

Soil-borne diseases of wheat, such as take-all and sharp eyespot, are important root diseases of wheat worldwide. No efficient control measures against soil-borne disease of wheat are available at present. Biological control of soil-borne disease by rhizobacteria, as an alternative measure, was intensively studied . Bacillus cereus which are able to form endospores that are tolerant to heat and desiccation, offer several advantages over other bacteria. Endophytic bacteria may not be affected by detrimental environmental factors against soil-borne pathogens have gained increasing scientific interest. .Proteolysis plays a critical role in maintaining cellular homeostasis by controlling the levels of various proteins, including global regulators involved in numerous biological functions. The ClpX belongs to the family of closely related Clp ATPases that function as molecular chaperones. Additionally, ClpX can interact with the related ClpP peptidase forming a proteolytic complex. In the ClpXP protease, ClpX serves to specifically recognize and subsequently unfold and translocate the substrate into the ClpP proteolytic chamber for degradation..B.cereus B3-7 is an endophytic bacteria, isolated from wheat roots, which have biocontrol capacity against soil-borne disease of wheat. we have constructed a transposon inserted mutants library contained 18000 strains. After screening in vivo, a biological control ability deficiency mutant acquired which was demonstrated clpX gene disrupted by transposon insertion. To elucidate the function of ClpX in the process of biological control. We constructed clpX deletion mutant（ΔclpX）with double crossover and homologus recombination. After testing bicontrol activity of B3-7,B3-7(ΔclpX) and B3-7(ΔclpX::TnYLB-1), we have revealed clpX is essential for biocontrol activities of B3-7. Total of 26 differential expression protein accumulated in clpX deletion mutant during the process of bicontrol toward soil-borne disease of wheat based on 2D electrophoresis were found. Based on research results previously, we would analyze the existence of ClpX recognition signal in 26 differential expression proteins after sequencing N-terminal and C-terminal amino acid with MALDI-TOF-MS and characterize ClpX specific recognition protein and its encoding gene. We then would carry out gene knock and compare the biocontrol activity against soil-borne diseases among mutants and its wt B3-7 so as to ascertain biocontrol related genes regulated by ClpX ATPase. Next ,we would analyze the metabolic circuits of biocontrol related genes involved and carry out multigene deletion to disclose temporal and spatial expression patterns of genes in the process of biocontrol. We would assay the stability of mRNA and its encoding proteins of biocontrol gene in wild type B3-7 and its derivative ΔclpX mutant to reveal the regulation patter caused by ClpX ATPase. All the research findings will lay a foundation of development biocontrol agents.

利用内生细菌防治病害，可以克服根际细菌生物防治中易出现的不稳定现象。研究内生细菌生防作用的调控网络，有助于深入理解生防作用机制。ClpX ATPase是广泛存在于各种生物细胞中的一种蛋白质量控制因子，参与调节细菌分化和抗逆性等多种基本生物学过程。前期工作我们证明了ClpX ATPase参与生防菌株蜡样芽孢杆菌B3-7对小麦全蚀病和小麦纹枯病的生物防治，但是受ClpX调控的生防因子类型和代谢网络有待阐明。本研究利用B3-7基因缺失突变体菌株ΔclpX在生防过程中发现的26个蛋白因子，鉴定受ClpX作用下表达差异蛋白的编码基因。通过基因敲除、蛋白互作分析并结合敲除菌株的生防能力测定，分离致病相关基因，解析生防基因参与的代谢网络。利用多基因突变分析，分析基因表达的时刻顺序。测定不同生防基因mRNA及其编码蛋白的稳定性，解析ClpX调控生防基因表达的方式，为进一步开发利用该生防菌株鉴定基础。

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