【作者】 李仙；

【导师】 朱菁萍；

【作者基本信息】 华中科技大学 ， 环境科学， 2011， 硕士

【摘要】 近年来,世界范围内蓝藻水华大量爆发,水华蓝藻产生的毒素已造成严重的污染和经济损失,蓝藻水华越来越受到关注。微囊藻是现有产毒蓝藻中的优势种。常规的蓝藻监测以显微镜下形态观察、计数为主,它要求操作者对各种蓝藻的细胞形态非常熟悉,否则易漏检或误检。因此,有待开发蓝藻尤其微囊藻的预警预测、分析方法。现代生物技术的发展以及基因数据库中海量的生物信息为我们建立以基因为检测对象、快速有效的检测方法提供了可能。PCR检测基因法已被报道,但它易产生假阳性。由于核酸分子杂交具有很高的灵敏度和高度的特异性,DNA探针—杂交方法可用于环境监测,此法更快速、灵敏。因此,本研究的目的是以微囊藻的PC-IGS序列为检测对象,建立一种水华蓝藻的预警预测的分子生物学方法,其主要工作内容和结论有:1.对微囊藻和鱼腥藻的PC-IGS序列进行比对分析并构建系统发育树,在PC-IGS系统发育树中,微囊藻和鱼腥藻形成两个独立的分枝,证实了PC-IGS序列在蓝藻种间具有高度的特异性,可用于建立检测蓝藻的分子生物技术。基于微囊藻的PC-IGS序列成功地设计、合成了对于微囊藻的检测具有较高特异性的捕获探针和信号探针。2.通过优化杂交液、杂交温度和时间等多项参数,建立了可以检测微囊藻的酶联夹心杂交方法。对实验室多株藻的杂交检测,验证了该法的可靠性和准确性,进而建立酶联夹心杂交方法标准曲线(y=0.00002x+0.1225,R2=0.9883)实现了水样的定量检测。该法的最低可检出浓度为1个/mL。3.利用酶联夹心杂交法、显微镜观察法和PCR法检测采集于武汉官桥湖、镜湖、源湖、喻家湖的实际水样,三者结果一致。显微镜观察计数的相对标准偏差会高达20.0%,而酶联夹心杂交法检测的相对标准偏差为14.1%。由此可见,酶联夹心杂交法是一种快速、有效的预警预测水华微囊藻的分子生物学方法。更多还原

【Abstract】 The increasing incidence of mass developments of cyanobacteria in fresh- and brackish water in whole-world is a matter of growing concern due to the production of toxins that threaten human and livestock health. Microcystis is the main producer of toxin-producing cyanobacteria. Technology for detecting cyanobacteria commonly use microscope to identify morphological characteristics, which requires the operator is very familiar with the cell morphology of various cyanobacteria, otherwise, it is easy to miss or false detect. Therefore, the accuracy of conventional microscopy is not high, and developing cyanobacteria, especially Microcystis early monitoring, analysis and early warning system is of great significance.The development of modern biotechnology and vast amounts of biological information in genetic database can offer the possibility of establishing rapid and effective methods to detect a target genotype. PCR method of detecting genes has been reported whereas it is easy to produce false. With respect to nucleic acid molecular hybridization’s obvious advantages of high sensibility and specificity, the conjugation of DNA probes with hybridization can be used for environmental monitoring, having advantages in high-speed and sensitivity.Therefore,the goal of the research is to establish a molecular biological method for early warning and forecast of Microcystis, which is based on PC-IGS sequence of Microcystis. In this thesis, the main contents and conclusions are as follows.1. The available PC-IGS (phycocyanin intergenic spacer) sequences are used for base comparison analysis and building phylogenetic tree. The Microcystis and Anabena strains form a monophyletic cluster respectively with respect to PC-IGS sequence. It is testified that PC-IGS sequence is conservative highly in cyanobacteria, and can used to establish molecular biological methods of detecting cyanobacteria. Pairs of high specific probes (capture probe and signal probe) are designed based on the PC-IGS sequence for the division Microcystis.2. The ingredients and concentration of hybridization solution, washing buffer, temperature, time of hybridization and so on hybridization parameters are optimized through many times tests, and the ELSHA method for monitoring Microcystis is established successfully. The correctness of the ELSHA was proved by the results of hybridization for Microcystis and Anabaena cultured in the laboratory. Then absorbance at 405nm of ELSHA is plotted against the cell concentration in the hybridization system to establish the calibration curve (y=0.00002x+0.1225,R2=0.9883) for ELSHA,which achieves quality detection for water samples. The minimal detectable concentration by ELSHA is only 1cells/mL.3. It is consistent that results of detecting the Microcystis concentration in water samples from GuanQiao Lake, Jing Lake, Yuan Lake, YuJia Lake by ELSHA, microscope and PCR. Further more, the relative standard deviation (RSD) by microscope examination maybe 20.0%, which is higher than 14.1% by ELSHA. Therefore, ELSHA developed in the study is a rapid and effective method for Microcystis bloom early warning and forecast.更多还原