Differential Expression Of Skin TLR MRNA Of RGV-stressed Rana Dybowskii

Toll-like receptor (Toll-like receptors, TLR) which can recognize pathogens and activate the immune response, is an important class of pattern recognition molecules involved in the innate immunity of the body, is also connected to the innate and acquired immunity bridge. The study of expression patterns to the molecular and pathogenic microorganisms can be a tremendous role in promoting the exploration of immunological. Amphibians skin as the tissue of directing contact with pathogenic microorganisms in their habitats, formed the first natural defense system against pathogenic microorganisms in the process of natural evolution, while TLR molecules have an important role in innate immune defense process. But the research about TLR is more concentrated in mammals, while the molecular mechanism of amphibians is unclear. Therefore, the use of quantitative PCR technique, the expression changes of TLR molecules in amphibians after pathogenic microorganisms infect over time, provide a scientific basis to reveal the amphibians activate the immune response mechanism in microbial invasion, and has a reference value to immunology research in amphibians.In this study, we selected Rana dybowskii which is amphibians representatives species as subject investigated, Rana grylio virus (RGV) as pathogenic microorganisms infected healthy adult R.dybowskii. We found abdominal organs by the anatomical lesions to confirm the frog iridescent virus infection. Selecting the frog skin tissue after the injection0,1,2,3,6,12,24,48,72,96,120,144,168and336hours as the experimental material, using SYBR Green I quantitative PCR technique, after optimizing the reaction system and reaction conditions, we detected TLRs molecule mRNA expression levels over the time, analysised the dynamic expression pattern, in order to explore if the Toll-like receptor involving in immune response after RGV infected, and the expression pattern of reaction with RGV.After designing primers and optimization of reaction conditions and reaction system, we successfully found quantitative PCR of the mRNA of TLR1, TLR2, TLR4, TLR5, TLR6and TLR8molecule in R.dybowskii skin. Using this method, the melting curve figure showing single specific peak, not existed specific amplification and primer-dimers, illustrated the optimal conditions provide a reliable quantitative method for the further study R.dybowskii skin gene expression after RGV stress. After RGV stress, the TLR mRNA relative expression levels in frog skin changes showed regularity, the peak of TLR1and TLR8mRNA were in1hour and6hours, the rest of TLR mRNA in0-2hours showed down-regulated, and in2-6 hours (the innate immune phase) increase and reached a peak, in particular TLR1, TLR4and TLR8were most significant. TLR2and TLR5were gradually raised in12hours (early induced response phase), and in72-96hours (acquired immune response stage) reached its peak. The control group of TLRs mRNA expression level showed stability, there were significant differences between the experimental group and control group (P<0.05).