甜瓜枯萎病拮抗细菌A6分泌有机酸耐铝的分子机制研究

About 21% of the arable land in China is acidic and in which aluminum toxicity is a problem affecting crop performance. Muskmelon is one of the important fruit crops, whose production is susceptible to Fusarium wilt because of aluminum toxicity in in acidic soils. The ability to colonize of microorganism antagonistic to the fungal pathogen is not effective, and it is necessary for the biocontrol microorganisms to be Al-tolerant where they are functioned in aluminum-toxic soils. The microorganisms antagonistic to Fusarium wilt of muskmelon that are also Al-tolerant are not well understood, and there is little information about the roles and mechanisms of proteins expressed by their genes. Based on the unsatisfactory biological effects, mainly caused by phytotoxic levels of aluminum in the acidic soil, the knowledge of both aluminum tolerance and the ability of microorganisms to antagonize the fungus are necessary for further studies. Therefore, we will use the methods of microbiology, molecular biology and bioinformatics to clone genes (clusters) related to secretion of organic acids, which is one of the main factors of aluminum tolerance. We will then explore the expression, enzyme features and mutation of the cloned gene clusters, confirm key active sites of the enzymes related to aluminum tolerance and clarify their mechanisms of action. This study will reveal the metabolic mechanisms of aluminum-tolerant A6 from the perspective of biochemistry and molecular biology, provide new gene resource and valuable information on genetic engineering of aluminum-tolerant microorganisms, and improve the aluminum tolerance and ability of A6 to colonize and antagonize Fusarium wilt of muskmelon in situ acidic soils.

我国酸性土壤所占面积大，铝毒严重；甜瓜是我国重要的水果作物，易受铝毒胁迫感染枯萎病，从而影响产量；甜瓜枯萎病主要利用生物手段防治，但拮抗菌不具有耐铝特性，定殖能力低，无法在铝毒土壤中起作用。目前耐铝甜瓜枯萎病拮抗菌未见报道，其耐受性分子机制未被明确揭示。本项目已筛选出分子操作相对简单、同时具有高效耐铝及防治甜瓜枯萎病两种功能的细菌A6，在明确其主要耐铝特征之一为分泌有机酸螯合铝的前提下，同时采用鸟枪法与底物显色相结合、转座子随机插入突变法及对菌株基因组序列进行信息分析，克隆其耐铝基因（簇）；探明野生菌及回复突变菌株在铝毒土壤中的定殖规律和拮抗效果，验证耐铝基因（簇）的稳定性；研究耐铝基因（簇）所表达蛋白质的作用机理，确定酶的关键活性位点，揭示菌株分泌有机酸耐铝的分子机制。本研究结果为获得性能优良的耐铝基因（簇），提高菌株在原位条件下的耐铝效果及定殖能力提供新的资源和思路。

酸性土壤中活性铝含量高，铝毒严重；而生防菌株要具备耐铝特性，才可成功应用于有铝毒的酸性土壤。目前耐铝细菌数量与种类较少，其耐铝机制未从基因角度被明确揭示。以耐铝甜瓜枯萎病拮抗菌株A6为主要研究对象，从生理生化、分子生物学及多组学三个角度互补验证，揭示了菌株分泌有机酸耐铝的分子机制。对菌株A6进行多相分类，进一步确定了其分类地位为Dyella sp.；测定了不同温度下铝离子对菌株A6分泌有机酸的影响，结果表明不同温度处理下相同量的菌株分泌物中，有机酸的主要种类（草酸、柠檬酸）及含量无明显变化，且外源柠檬酸、草酸对菌株有较为明显的铝毒缓解作用；利用转座子随机插入突变法、基因组学、转录组学、蛋白组学测序联合阐述了菌株A6耐铝代谢网络，并确定研究重点为三羧酸循环通路，克隆出异柠檬酸脱氢酶、苹果酸脱氢酶基因，通过回复突变与原核表达验证了其功能，并研究了其表达蛋白的酶学特性及作用机理；改良了菌株A6的生长培养基，分离及鉴定了6株高效耐铝功能菌，为铝毒复合污染土壤提供了改良材料；阐明了耐铝甜瓜枯萎病拮抗菌在甜瓜根系、含铝根际土壤及各种固体载体中的定殖规律及拮抗效果，菌株在甜瓜根系、含铝土壤中定殖能力较高，且可随着有机肥的施用发挥更好的应用效果。本研究揭示了菌株A6分泌有机酸耐铝的分子机制，获得了性能优良的耐铝基因，为铝毒严重的酸性土壤改良提供了良好的菌种、基因资源和理论基础。