【作者】 王凯；

【导师】 闫培生；

【作者基本信息】 哈尔滨工业大学 ， 环境科学与工程， 2013， 博士

【摘要】 环境中存在多种生物致癌因素，如黄曲霉在土壤中广泛存在，在适宜条件下侵染寄生体产生毒素。黄曲霉毒素能引起人畜的急慢性中毒，且具有“三致”作用，甚至影响生态安全。花生在我国是重要的油料和经济作物，但黄曲霉毒素含量超标非常严重。传统的方法是利用农药防治，但农药的大量频次使用将造成农业面源污染，对土壤、水体甚至大气产生危害。对环境友好的生物防治技术是花生产前治理黄曲霉毒素污染的有效方法，而微生物农药是生物防治的重要手段。本研究利用不同类型的培养基，从全国6个省份花生主产区的花生样品果壳中分离获得内生细菌236株。经过可视化平板培养筛选，其中72株对黄曲霉毒素产生有明显抑制效果。采用tip-culture方法定量确定了其中24株对菌丝生长的拮抗率大于90%；54株对黄曲霉毒素产生的拮抗率大于90%。以16S rDNA基因序列分析为基础，结合形态学观察和部分生理生化特性测定，将筛选出的72株内生菌进行系统发育学分析。结果表明，归于芽孢杆菌科（Bacillaceae）的绝大部分（48株）属于芽孢杆菌属（Bacillus），为具有生防作用的花生果壳内生菌的优势种群，分别聚类于7个种：解淀粉芽孢杆菌（B.amyloliquefaciens），枯草芽孢杆菌（B. subtilis），甲基营养型芽孢杆菌（B.methylotrophicus），短小芽孢杆菌（B. pumilus），特基拉芽孢杆菌（B.tequilensis），梭状芽孢杆菌（B. fusiformis）和蜡样芽孢杆菌（B. cereus）；归于活动球菌科（Planococcaceae）的仅有一株BPM12-1，与Lysinibacillus xylanilyticus CCUG57438T亲缘关系最近。归于肠杆菌科（Enterobacteriaceae）的18株分别属于肠杆菌属（Enterobacter）和沙雷氏菌属（Serratia），其中肠杆菌属（Enterobacter）的7株分别属于阿氏肠杆菌（E.asburiae）、阴沟肠杆菌（E.cloacae）和E.ludwigii；沙雷氏菌属（Serratia）的11株均为粘质沙雷氏菌（S. marcescens）。归类于黄单胞菌科（Xanthomonadaceae）的3株属于寡养单胞菌属（Stenotrophomonas）的嗜麦芽窄食单胞菌（S. maltophilia）。归类于假单胞菌科（Pseudomonadaceae）的2株属于假单胞菌属（Pseudomonas）的地中海假单胞菌（P. mediterranea）。采用透明圈法确定筛选出的72株内生菌降解几丁质的能力，其中22株有水解圈产生。采用DNS法测定其中各类群代表菌株的酶活，沙雷氏菌属（Serratia）和寡养单胞菌属（Stenotrophomonas）的代表菌株酶活相对较高。选择其中一株酶活较高且生防作用显著的细菌，综合利用表型、生理生化特征、基因型和系统发育分析结果将该菌鉴定为粘质沙雷氏菌（Serratia marcescens），菌株编号为JPP1。通过PCR扩增获得长度为1789bp的核苷酸序列，含有一个完整的1500bp的开放阅读框，编码499个氨基酸，经过相似性比对和系统进化分析，确定菌株JPP1所产几丁质酶为ChiB。该酶分子量为55480.3Da、等电点5.93、较稳定、属脂溶蛋白、具有一定亲水性。二级结构为31.66%的α螺旋，20.64%的延伸带和47.70%的无规则卷曲。三级结构与一株野生型粘质沙雷氏菌所产几丁质酶ChiB复合物的结构同源性为98.6%。通过PB实验设计和中心组合设计法确定粘质沙雷氏菌JPP1的产几丁质酶优化培养基配方为：胶体几丁质12.70g，葡萄糖7.34g，蛋白胨5.00g，硫酸铵1.32g，K2HPO₄0.7g，MgSO₄·7H2O0.5g。通过中心组合设计法，确定发酵条件的装液量、转速、接种量分别为23.2ml、116rpm和4.3%时，所建立模型的最大响应值酶活为31.96U，摇瓶培养验证实验结果与之较为接近。几丁质酶的稳定性试验显示该酶热稳定性较好；pH值5⁸范围内相对酶活在80%以上；Zn²⁺、Cu²⁺、Fe3+对酶活有明显的抑制作用，Ca²⁺、Mn²⁺对酶活有一定的促进作用，Na+和K+对酶活的影响不大；保护剂EDTA、巯基乙醇和Tween80可以一定程度保护酶的活性，变性剂SDS则使得酶活有明显降低。扫描电镜结果显示菌株JPP1产生的几丁质酶使得寄生曲霉菌丝的细胞壁被部分降解；采用tip-culture方法，确定酶液浓度与菌丝生长的抑制率和毒素产生的抑制率呈现出良好的线性关系；TLC实验结果显示经酶液处理后产毒寄生曲霉菌株没有明显产生黄曲霉毒素。RT-PCR实验表明经过该酶处理的曲霉菌株不存在aflR、aflC（pksL1）和aflO（dmtA）基因的转录。因此确定该菌株产生的几丁质酶是其发挥生防作用的主要机制。确定花生生物种衣剂的主要配方，配比为4:1的聚乙烯醇和羧甲基纤维素钠为复合成膜剂，增塑剂为1%的甘油，与发酵菌液按照4:1的配比混合均匀后包衣，再用2%的CaCl2作为交联剂处理。该配方对生防粘质沙雷氏菌JPP1的生长没有抑制作用。花生种子包衣处理后，发芽率达到98%，且对种子萌发有明显的促进作用；实验室中对毒素产生的抑制率达到88.5%，优于农业常用杀菌剂多菌灵的抑制效果。更多还原

【Abstract】 A variety of biological carcinogens exist in the environment，for exampleAspergillus flavus is widely present in the soil and infects the host under appropriateconditions to produce aflatoxins （AF）. AF can cause acute and chronic poisoning,but also has carcinogenic, teratogenic and mutagenic effects on humans and animals,even could affect ecological safety. Peanut is an important oil and economic crop inChina， but the aflatoxin content in peanut exceeds the standard seriously.Traditional control method was to use chemical pesticides, but a large number ofhighly toxic pesticides continuous use will result in agricultural non-point sourcepollution and harm to the soil, water and atmosphere. Biological control technologyis friendly to the environment and effective to control AF contamination in peanuts.Microbial pesticide is one of the important means of biological control.Using several kinds of media,236strains of endophytic bacteria weresuccessfully isolated from hulls of peanut plants growing in6provinces in China.By visual agar plate assay to screen,72strains of endophytic bacteria significantlydecreased AFs production. Using tip-culture method24strains were determined tohave decreased mycelia growth levels by>90%and54strains decreased AFsproduction levels by>90%.A total of72isolates of endophytic bacteria were sequenced for their16SrDNA，combined with morphological observation and some physiological andbiochemical characteristics to analyze their phylogenetic relations. The majority ofendophytic bacteria in Bacillaceae belonged to the genus Bacillus （48），which wasdominant and could be classified into seven species, B.amyloliquefaciens, B.subtilis, B. methylotrophicus, B. pumilus, B. tequilensis, B. fusiformis and B. cereus.The only one strain BPM12-1affiliated with the family Planococcaceae, andexhibited99%similarity with the strain of Lysinibacillus xylanilyticus. There were18strains affiliated with the family of Enterobacteriaceae with the two genera ofEnterobacter and Serratia. Among them,7strains belonging to the genus ofEnterobacter could be classified into three species, E. ludwigii, E. asburiae, and E.cloacae. The other11strains belonging to the genus of Serratia were related to thesame species Serratia marcescens. There were3strains which affiliated with thefamily of Xanthomonadaceae belonged to the same genus of Stenotrophomonas andshowed sequence similarity value of99%to Stenotrophomonas maltophilia. Thetwo strain affiliated with the family Pseudomonadaceae and exhibited99% similarity with the strain of Pseudomonas mediterranea.By transparent ring method22strains of identified endophytic bacteriadegraded chitin on the chitin medium plate. Using DNS method to determine theenzyme activity of represent bacteria, the strains which showed stronger chitinolyticactivity belonged to the genera Serratia and Stenotrophomonas. A strain with higherenzyme activity and exhibited remarkable biocontrol effect was selected. Combinedwith morphological, physiological, biochemical characteristics and phylogeneticrelations, the strain was identified as Serratia marcescens and designated S.marcescens JPP1. Polymerase chain reaction （PCR） amplification of the chitinasegene was performed and obtained a1,789bp nucleotide sequence, its ORF was1,500bp and encoded499amino acids. After similarity alignment and phylogeneticanalysis, the chitinase produced by strain JPP1was determined as ChiB. Themolecular weight and the isoelectric point of ChiBjp were55480.3Da and5.93,respectively. It was a fat-soluble protein and relatively stable and hydrophilic. Itspredicted secondary structure was31.66%alpha helix,20.64%extended strand and47.70%random coil. Its predicted tertrary structure exhibited similarity of98.6%with ChiB from S. marcescens wildtype in complex with catalytic intermediate.Using Plackett-Burman design and central composite design, the mediumformula for chitinase production of S. marcescens JPP1was optimized. The formulawas （g/l）: colloidal chitin12.70, glucose7.34, peptone5.00,（NH4）2SO₄1.32,K2HPO₄0.7, MgSO₄·7H2O0.5. By central composite design, the maximumresponse enzyme activity of established model was31.96U when the fermentationconditions of volume, rotary speed and inoculum size were separately23.2ml,116rpm and4.3%. The result of flask fermentation experiment was close to themaximum enzyme activity.Stability test of chitinase exhibited better thermal stability and the relativeenzyme activity was more than80%at pH5⁸. The metal ions of Zn²⁺, Cu²⁺andFe3+showed significant inhibitory effect on the chitinase activity, while Ca²⁺andMn²⁺had positive effect, Na+and K+had little effect on chitinase activity. Proteinprotectants of EDTA, mercaptoethanol and Tween80could protect the chitinaseactivity in certain degree, while denaturant of SDS significantly reduced thechitinase activity.SEM analysis showed major damage of the mycelia and the cell wall of A.parasiticus was partially degraded in the presence of chitinase produced by S.marcescens JPP1. Using tip-culture method the chitinase concentration withantifungal ratio and antiaflatoxigenic ratio exhibited satisfied linear relationship.TLC experiment showed that toxigenic A. parasiticus treated with chitinase had not visibly produced AFs. RT-PCR results showed that ChiBjp repressed thetranscription of aflR, aflC （pksL1） and aflO （dmtA） genes. Therefore, the chitinaseproduced by the strain played the biocontrol effect.The formulation of biological seed coating agent was determined, polyvinylalcohol and carboxymethyl cellulose sodium （4:1, v/v） were mixed to yield thecomplex filmogen. The glycerin （1%） acted as plastifier, then the whole bacteriummedium and the filmogen （1:4, v/v） were fully mixed. After coating, the peanutseeds were treated with CaCl2（2%） as crosslinker. The formulation did not inhibitthe growth of biocontrol bacterium. After coating treatment, the germination rate ofpeanut seeds was up to98%. The seed coating agent significantly promoted the seedgermination and in vitro the antiaflatoxigenic ratio was up to88.5%, its antagonisticeffect was better than the agricultural fungicide of carbendazim.更多还原