莠去津矿化群落NC1的筛

发布时间：2018-05-03 02:21

  本文选题：莠去津 + 矿化　； 参考：《东北农业大学》2016年硕士论文

【摘要】：莠去津又名阿特拉津,英文名:Atrazine,属于三嗪类除草剂,在过去的40年中已被广泛应用于农作物的杂草控制,如高粱、玉米和甘蔗。因为长期大范围使用目前已造成了严重的环境污染,在作物生长的土壤、水体中均已检测到莠去津存在,它不仅破坏生态环境,还严重危害着人类的健康。本论文的研究目的是筛选出污染土壤中的莠去津矿化群落,并进行分离鉴定。在国内外微生物矿化莠去津研究的基础上对这些矿化微生物的矿化特性进行较为系统的研究。本研究利用富集培养法,从黑龙江省伊春市南岔区长期施用莠去津的玉米地中筛选出莠去津高效矿化群落NC1,此群落可以在28 h时矿化MSM培养基内99%的100 mg/L的莠去津,使其转变为无毒的氰尿酸。通过MSM平板直接分离法,分离出5株形态、颜色各不相同的菌株。通过生理生化和16S rDNA鉴定,菌株YJY1、YJY2、YJY3、YJY4和YJY5分别被鉴定为肠道杆菌(Enterobacter sp.)、无色杆菌(Achromobacter sp.)、铜绿假单胞菌(Pseudomonas sp.)、希瓦氏菌(Shewanella sp.)和克雷白氏肺炎菌(Klebsiella sp.)。并将上述五种菌株分别命名为Enterobacter sp.YJY1、Achromobacter sp.YJY2、Pseudomonas sp.YJY3、Shewanella sp.YJY4和Klebsiella sp.YJY5。底物利用实验及矿化实验表明菌株YJY4和YJY5可以以莠去津为唯一氮源生长,所有群落成员均不能利用氰尿酸为唯一氮源生长,且菌株YJY4矿化莠去津效率明显强于菌株YJY5,所以确定菌株YJY4为矿化菌落NC1的主要成员。菌株YJY4是第一个被发现可以矿化莠去津的希瓦氏菌(Shewanella sp.),这表明它对莠去津污染的土壤环境的修复具有巨大潜力。在此基础上,我们对YJY4降解菌的特性进行了研究。根据对已发表的莠去津矿化微生物基因序列设计引物,通过PCR技术对于菌株YJY4基因组进行扩增,在菌株YJY4中分别发现了莠去津矿化基因atz A、atz B和atz C基因,通过序列比对,发现扩增出的三种矿化基因与已发表的假单胞菌ADP和节杆菌TC1中的矿化基因相似性很高,达到了98%以上,这也证明了菌株YJY4可以将莠去津矿化成无毒无害的氰尿酸。利用定时培养,每隔4 h测定OD600nm处吸光值观察菌株生长情况,证实菌株YJY4在近似中性的pH(6.0-8.0)范围内都可以很好生长,其最适碳源和氮源分别为蔗糖和硝酸铵。
[Abstract]:Atrazine, also known as atrazine, is a triazine herbicide and has been widely used in crop weed control in the past 40 years, such as sorghum, corn and sugar cane. Because the long-term extensive use has caused serious environmental pollution, atrazine has been detected in the soil and water body of crop growth, which not only destroys the ecological environment, but also seriously endangers human health. The purpose of this paper is to screen out the mineralized communities of atrazine in contaminated soil and to separate and identify them. On the basis of the research of microbial mineralization atrazine at home and abroad, the mineralization characteristics of these mineralized microorganisms are studied systematically. In this study, the enrichment culture method was used to screen atrazine highly effective mineralized community NC1 from the corn field of Nancha District, Yichun City, Heilongjiang Province, which could mineralize 99% of Atrazine 100 mg/L in MSM medium at 28 h. Turn it into a nontoxic cyanuric acid. Five strains with different color and morphology were isolated by MSM plate method. By physiological and biochemical identification and 16s rDNA identification, the strains YJY1, YJY2, YJY3, YJY4 and YJY5 were identified as Enterobacter sp., Achromobacter sp., Pseudomonas sp. and Shewanella sp., respectively. And Klebsiella sp. The above five strains were named as Enterobacter sp. YJY1Achromobacter sp. YJY2 Pseudomonas sp. YJY3 Shewanella sp.YJY4 and Klebsiella sp. YJY5. The substrate utilization and mineralization experiments showed that YJY4 and YJY5 could grow with atrazine as the only nitrogen source, and all the community members could not use cyanuric acid as the sole nitrogen source. The mineralized atrazine efficiency of strain YJY4 was significantly higher than that of strain YJY5, so strain YJY4 was the main member of mineralized colony NC1. Strain YJY4 is the first strain of Shewanella sp. found to be able to mineralize atrazine, which indicates that it has great potential for remediation of atrazine contaminated soil environment. On this basis, we studied the characteristics of YJY4 degrading bacteria. According to the published gene sequence of atrazine mineralized microorganism, primers were designed and amplified by PCR technique. The genes of atrazine mineralization gene atz Agnatz B and atz C were found in strain YJY4, respectively. It was found that the three mineralized genes were similar to the mineralized genes of Pseudomonas pseudomonas ADP and Arthrobacter TC1, reaching more than 98%, which proved that the strain YJY4 could mineralize atrazine into innocuous cyanuric acid. The absorptivity of OD600nm was measured every 4 hours. The results showed that YJY4 could grow well in the range of pH 6.0-8.0). The optimum carbon source and nitrogen source were sucrose and ammonium nitrate, respectively.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：X592;X172
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本文编号：1836545