The Detection Of Genes Encoding Enzymes From Microorganism In Compost System Using The Technology Of Biosensor And Real-time Quantitative PCR

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.