【作者】 罗洁；

【导师】 曾光明；

【作者基本信息】 湖南大学 ， 环境工程， 2011， 硕士

【摘要】 随着社会发展,农业废物的无害化、减量化和资源化处理,越来越受到人们的重视。通过合理的堆肥可以实现这一目标。木质素、纤维素是农业废物堆肥化过程中的主要限速有机物,其降解被认为是快速堆肥的关键。黄孢原毛平革菌在降解木质素方面有着重要意义,本研究以黄孢原毛平革菌的基因组DNA为模板,根据木素过氧化物酶的基因序列设计引物,比较了此菌基因组DNA的提取方法,并通过聚合酶链式反应和酶切反应得到339bp及265bp的待测特异性DNA片段,制作DNA传感器以检测所得DNA片段。利用自组装单分子膜法,将巯基修饰的探针固定在金电极表面,并研究了夹心式和竞争式两种杂交机制,采用辣根过氧化物酶作为标记物。在电化学分析中采用了循环伏安法、交流阻抗法、方波伏安法、微分脉冲伏安法等一系列电化学分析方法。研究表明:SDS-CTAB法提取黄孢原毛平革菌基因组DNA效果较好。电化学分析中,电流响应值与DNA检测浓度成线性关系。夹心式杂交机制中,线性回归方程为y=8×1010x+0.280,线性相关系数为0.981,回收率为97.93%～113.53%。竞争式杂交机制中,作出生物传感器检测黄孢原毛平革菌DNA低浓度段曲线,回归方程为y=-4×1010x+0.366,线性相关系数为0.993。其中y为电流跃迁值(μA);x为DNA检测链浓度(mol/mL)。此外,对传感器进行再生,实验结果表明再生性能较好。纤维素的降解是堆肥中很重要的一个过程,产纤维素酶能力最强的常用的微生物是木霉属的里氏木霉。本研究采用DNA生物传感技术以及实时荧光定量PCR技术对其能产生纤维二糖水解酶的编码基因进行检测,达到检测堆肥进程的目的。DNA初始浓度的对数值与循环阈值成良好的线性关系,回归方程为y=-2.7563x+38.284,r2=0.9903,达到了较好的效果。更多还原

【Abstract】 With the development of the society, more and more attention has been paid to realizing the decontamination, reduce and recycle of the agricultural waste, and composting is a widely accepted way. Lignin and cellulose as organic matter hardly to be degraded in compost are considered as the key point of fast compost. Phanerochaete chrysosporium is of great significance in the degradation of lignin. The genome DNA of Phanerochaete chrysosporium was studied as a template and the primers were designed according to the lignin peroxidase gene sequence.We have compared the methods of DNA extraction and got 339 bp and 265 bp specific DNA fragments which were amplicated by polymerase chain reaction and restriction enzyme digestion. The DNA sensor was made to detect the DNA fragments.The Mercapto-modified probe was immobilized on the surface of gold electrode with the method of self-assembling single-molecular film.The horseradish peroxidase was adopted as a marker in the two hybridization mechanism of sandwich and competition. Electrochemical analysis such as cyclic voltammetry, electrochemical impedance spectroscopy, square-wave voltammetry and differential pulse voltammetry were used. Research showed that the method of SDS-CTAB to extract the DNA of Phanerochaete chrysosporium is the best. The DNA sensor displayed an excellent electrical response linear to the reduction of DNA concentration. As for the sandwich hybridization of DNA sensor, the linear regression equation is y=8×1010x+0.280, with a correlation coefficient of 0.981, and the recoveries are in the range of 97.93%~113.53%. And the linear regression equation of hybrid-competition mechanism is y=-4×1010x+0.366, in the range of low DNA concention, with a correlation coefficient of 0.993, where y is current transition value,μA, and x is DNA concentration, mol/mL. It shows that the regeneration of the sensor has a good performance. The degradation of cellulose is one of the most important steps in the process of compost.Trichoderma Reesei in the trichoderma has high ability to produce cellulase. This research detected the DNA of cellobiohydrolase which is produced by Trichoderma Reesei depending on the technology of DNA biosensor and real-time quantitative pcr, and it achieved the aim of detecting the process of compost. The standard curve indicated the linear relationship between cycle threshold template concentration and showed high correlations (r2= 0.9903), and the linear regression equation of is y=-2.7563x+38.284.更多还原