Construction Of Detection Method For Transgenic Plants Based On RNAi Technology And Research On Signal Amplification Strategy

RNA interference(RNAi)refers to the phenomenon of m RNA degradation caused by an internal or exogenous double-stranded RNA(ds RNA),resulting in the specific blocking of target gene expression.RNAi induced gene silencing has been widely used in functional gene research,gene expression regulation,development of new pesticides,pest control and other fields due to its high efficiency,specificity,rapidity and simplicity.In recent years,RNAi mechanisms have also been used to develop transgenic crops with superior traits and antiviral ability.With the in-depth exploration of plant genome and the discovery of gene editing tools by scientists,transgenic plant technology has developed rapidly.However,the production and distribution of genetically modified plants has also raised concerns about biosafety.Although countries around the world are constantly improving relevant laws and regulations on regulating the management of GM products,there is still no safe and unified management system due to regional restrictions,threshold range,labeling system and other factors.Therefore,the detection of exogenous ingredients in introduced transgenic products has always played an important role in the evaluation of genetically modified organisms.The content of exogenous DNA introduced by transgenic technology is usually low.Signal amplification strategy is adopted to meet the analysis requirements of actual samples when the detection system is established.The introduction of exogenous DNA into the construction of the detection system can significantly improve the analytical performance and reduce the detection limit,which provides a new research idea for the assembly and application of electrochemical DNA sensors.In this paper,electrochemical DNA sensor and CRISPR technology were applied to the detection and analysis of RNAi related transgenic plants in combination with different signal amplification strategies.The main research contents are as follows:(1)An electrochemical sensor based on DNA probe hybridization was constructed for quantitative detection of long double stranded RNA in transgenic maize leaves.A novel Tri-block DNA capture probe was designed and the surface of the gold electrode was modified.In order to reduce the unnecessary nonspecific adsorption on the electrode surface,6-mercaptohexanol was used to seal the binding site on the electrode surface before hybridization.Electrochemical signals were monitored by chronoamperometry.The successful modification of the electrode interface and probe hybridization were characterized by X-ray photoelectron spectroscopy(XPS).After a series of optimization conditions,we could detect the synthetic RNA as low as 10 f M,and detected the specific sequence in 3637.5ng/ ?l total RNA extracted from transgenic maize leaves.The quantitative analysis of transgenic double-stranded RNA extracted from actual samples was carried out by PCR method,which verified the practicability of the sensor.Therefore,the electrochemical DNA sensor proposed in this work opens up a new way for gene sensitive detection of transgenic plants.(2)A DNA hybridization detection system based on clustered regularly interspaced short palindromic repeats(CRISPR)system and recombinase polymerase amplification(RPA)was developed to realize the fluorescence quantitative and sensitive detection of nucleic acids in transgenic plants.In this experiment,the target sequence was amplified by one-step RT-PCR,and the sequence specific c RNA was designed to guide CRISPR-cas13 a to directly detect ds RNAs and activate their non-specific cleavage activity.When the signal molecules labeled with fluorescent groups were introduced into the detection system,fluorescence signals can be observed through the fluorescence spectrophotometer to detect specific wavelengths,so as to realize sensitive detection of targets with different concentrations.Under the optimal conditions,the detection range of the method is 100 a M-10 p M,and the minimum detection limit is 100 a M.In addition,the method shows good selectivity,excellent practicability and short analysis time in the actual sample detection,which has broad prospects in clinical analysis.