BSA Protein Molecule Detection Based On Solid-state Nanopores And The Research Of Synthetic Gene About Citrinin And Monacolin K In Monascus Spp

Nanopore widely attracted scientists attention as one of the most potentialmethods in the highly sensitive sensor field, so the solid-state nanopore sensor hasbecome the general method in the detection and analysis of the singlemolecules.Except using in DNA detection, the nanopore technology has been appliedin diverse biological macromolecular samples such as protein molecules andnanoparticles.Nanopore is prepared and assembled on the thin film, which size isapproximately10-1~102nm. In electrolyte solution and the applied bias voltage on theboth sides of the pore film, the charged particles would go through the nanoporeunder the effect of electric field force. This process of the charged samples goingthrough the nanopore channel called translocation. Translocation behavior will causethe instantaneous ion current changes, which are characterized as short-term spikes.Each peak corresponds to a translocation event. Through the analysis of the currentchange and translocation time, we can analyze the particle size, surface properties andtheir motor behaviors.Monascus is a kind of important filamentous fungi, which can produce bioactivesubstances. It can be used as a food additive and some cholesterol-lowering drugs, butat the same time the strains can produce citrinin, it is harmful to the human, thereforecontrolling the content of citrinin is the key of the research on Monascus. This articleis using the gene knockout technology to study the citrinin synthesis related genes,which provides the reference to understand metabolic pathways of citrinin inMonascus.This topic mainly includes two parts. The first part is using bovine serumalbumin (BSA) and gold nanorods (AuNPs) as samples to research the theirtranslocation and data analysis for nanopore sensor in protein research.The secondpart is using PCR amplification and hybridization method based on laboratoryexisting34Monascus strains to conduct the citrinin and Monacolin K synthesisrelated gene, finally we verified ctnF gene disruption mutants in Monascus sppAs3.4384and use HPLC method to detect the secondary metabolites citrinin content,which provides reference for the research about monascus strains. The maincontents are as follows:The first part:1. We used the silicon nitride nanopores chips to preliminary to study thetranslocation behavior of the BSA and gold nanorods. Finally we successfullyestablished the method using the nanopores to detect protein samples andnanoparticles.2. We carried out the statistics and analysisthe for the translocation signals ofprotein molecules and nanoparticles. We established the molecular translocationmotion model and calculated the results according to the actual translocationexperiment condition parameters, which were in accordance with the experimentalresults, eventually realizing the molecular real translocation events analysis.According to the translocation signals from the experiment, we summarized thecommon type of translocation signals.3. According to the experimental results, we simulated the electric field of theinner and around of the solid-state nanopores to get the comparison of the electricfield intensity about the same nanopores under different voltage. The bigger was thevoltage, the stronger was the electric field distribution around the pore.The fasterwere the molecules moving, the more were the molecular translocation.This topic aims to explore the silicon nitride nanopore application in proteindetection, which provides experimental and theoretical basis for subsequentnanopores sensing in protein research.The second part:1. We activated the laboratory existing34Monascus strains, extracted genomeDNA as templates and designed the amplification primers to conduct the citrininsynthesis related genes and the Monacolin K synthesis related gene, and verified thesynthetic genetic mokB and regulate gene mokH of Monacolin K by DNA dothybridization.2. According to the large subunit ribosomal RNA (LSU) D1/D2area, we usedthe oligo software to design primers for PCR amplification, then to sequencing theamplification products. We got that the A base in about71bp location of strain5and strain6different with the G base of other species in this position. The result and theamplification results about citrinin synthesis related genes that indicate strain5andstrain6belong to red Monascus.3.To verify the gene disruption mutants about transformants which have beentransfered ctnF gene targeting vector, and compare the secondary metabolites ofcitrinin and monascus pigment between the original strain As3.4384and genedisruption mutants, we found the content of citrinin in ctnF gene disruption mutantsis lower about34%than the original strain; The production of monascus pigmentreduce72%, so we could confirm ctnF gene is associated with the synthesis ofmonascus pigment and citrinin.This topic aims to research the existing monascus strains in molecules, whichprovides the experimental basis for subsequent further molecules studies aboutmonascus strains.