Investigation Of The Molecular Mechanism Underlie The Rapid Development Of C. Elegans Induced By Electromagnetic Fields Using High-through Sequencing Technology And The Dirving Force Behind The Overall Inverse-s Shaped Curve Of The Ampliifcation Efficienc

As the rapid development of communication technology as well as the wideapplication of electric power and household electric appliances, electromagnetic fields(EMF) in the environment rises sharply and harms to people. Epidemics surveys showthat EMF may cause the dysfunction of nervous system, cardiovascular system,immune system as well as genital system.Experimental evidences reveal that injurybiological effect such as thermal effect and non-thermal effect can be induced byEFM.Therefore, study on the bio-effect as well as molecular mechanism caused byEMF has been the hot point in the field of Electromagnetism Biology. In the past years,study on the bio-effects caused by EMF made a great progress. However, themolecular mechanism underlie the bio-effect induced by EMF was still unclear. Itslows down the development of medicine for the injury induced by EMF andinfluences the establishment of protective standard and measurement. As a result,molecular mechanism behind the bio-effect caused by EMF has been an urgentquestion. In order to have a deep insight into the molecular mechanism underlie thebio-effect caused by EMF, C.elegans was applied as experimental model to investigatethe bio-effect induced by EMF in our lab. Primary findings reveal that the growth rateof C.elegans was significantly accelerated by EMF. In this study, High-throughSequencing technology was applied to screen target genes and signal pathways respondto EMF to reveal the molecular mechanism behind the rapid development induced byEMF. We hope that our findings could provide new clues to the study on the molecularmechanism underlie the bio-effects caused EMF. In addition, when Q-PCR and duplexPCR were applied to identify the results of High-through Sequencing technology, wefound that amplification efficiency (Ea) was an important parameter which has a closerelationship to the application of PCR. Therefore, we investigate the driving force behind the overall inverse-S shaped profile of the amplification efficiency decline. Themain results were as follows:1. Investigation of molecular mechanism underlie the rapid development ofC.elegans induced by electromagnetic fields (EMF) using high-throughsequencing technologyObjects: To screen target genes and signal pathways respond to the developmentacceleration of worms induced by EMF as well as to check whether the developmentacceleration of worms was partly ascribed to non-thermal effect of EMF by usingHigh-through SequencingMethods: Four different samples labeled as wave-36h,25℃-36h,ctrl-48h and ctrl-36hwere collected for performing High-through Sequencing according to the growth curveof worms in wave exposure group(1750MHz,2.5W,20℃), control group(20℃,without EFM) and temperature control group(25℃, without EFM), respectively. Then,data was analyzed by bioinformatic method to identify differential expressed genes andmicro-RNA between wave-36h and25℃-36h samples as well as to identifymicrowave-response genes in wave-36h sample. In addition, microRNA regulatorynetwork associated with microwave exposure was established.Results:372up-regulated genes,18down-regulated genes,20up-regulatedmicroRNA and16down-regulated microRNA were found between wave-36h andtemperature control25℃-36h sample. It was found that630genes were specificallyexpressed in wave-36h sample. They were involved in biological processes linked tomolting cycle, body morphogenesis and cuticle development. In addition, genesinvolved in hedgehog signal pathway such as grl-5, grl-7, wrt-1, ptr-18, ptr-16, wrt-2and grd-1were significantly up-regulated in wave-36h and negative controlled bymiR-230, miR-56, miR-57.Conclusion: The rapid development of C.elegans induced by EMF is partly ascribed to the non-thermal effect of EMF. EMF accelerates the development of C.elegansperhaps by promoting the expression of collagen gene and cuticle gene.2. Combination of the duplex PCR technology with capillary electrophoresis isan alternative method to validate the result of transcriptome sequencingObjects: To establish a method to validate the result of transcriptome sequencing byusing duplex PCR technology combined with capillary electrophoresis.Methods: According To previous study of the transcriptome sequencing, eightdifferentially expressed genes were chosen as target genes for examination. The mRNAexpression level of these genes was detected using duplex PCR combined with agarosegel electrophoresis, duplex PCR combined with capillary electrophoresis and Q-PCR,respectively. Then, the verification efficiency of each method was evaluated carefully.Results: The verification efficiency of duplex PCR combined with agarose gelelectrophoresis was50%, while the verification efficiency of duplex PCR combinedwith capillary electrophoresis and Q-PCR both were100%.Conclusions: Combination of the duplex PCR technology with capillaryelectrophoresis can be used as an alternative method to validate the results oftranscriptome sequencing.Conclusion: Combination of the duplex PCR technology with capillaryelectrophoresis can be used as an alternative method to validate the results oftranscriptome sequencing.3. Investigation of the driving force behind the overall inverse-S shapedcurve ofthe amplification efficiency in PCR reactionObjects:To explore why the amplification efficiency decreases in the forms of inverseS-shaped curve in PCR reactionMethods: First, renaturation efficiency (Er) was introduced to indicate the efficacy of template renaturation reaction (TTR). Second, Q-PCR was conducted to explore howrenaturation efficiency changes during the course of PCR. Third, that the competitionbetween PCR and TRR during the whole reaction was analyzed from the view ofthermodynamics was used to judge whether TRR is the main factor contributing to theattenuation of amplification efficiency in the form of inverse S-shaped profile. Fourth,Q-PCR was performed to investigate how template concentration influences the profileof amplification efficiency as well as renaturation efficiency. Result was used asevidence to check whether theoretical analysis above is right or not. Finally, otherfactors such as primer depletion, dNTP exhaustion, heat inactivation of DNApolymerase and enzyme saturation were also investigated to identify whether they playan important role in the decay of amplification efficiency by investigating the reasonslead to the plateau phase of PCR.Results: Renaturation efficiency increases in the forms of overall S-shaped profileduring the course of PCR. The result of theoretical analysis and experiment shows thatTRR is the main driving force behind the overall inverse-S shaped profile of theamplification efficiency decline. At the plateau phase of PCR, primer and dNTPdoesn’t used up, enzyme keep high activation and templates renaturation happened. Asa result, none of primer depletion, dNTP exhaustion, heat inactivation of DNApolymerase and enzyme saturation was the main factor contributed to the decay ofamplification efficiency.Conclusion: Template reannealing is the main factor contributed to the decay ofamplification efficiency in the forms of inverse “S” shaped curve as well as to theattainment of plateau in PCR.