| Taking the Rana dybowskii as the research object;and taking the Escherichia coli and Staphyloccocus aureus as the differential origin of stimuli,total RNA was extracted from back skin with modified Triol method(48 hours after dead bacterial stimuli Rana dybowskii).Then methods Two-step RT-PCR was utilized for cDNA synthesized,and in this study the upstream primer(Sense) was designed according to the n conservative region of skin antibacterial peptide(ABP) genes with Rana genus;and the downstream primer is Oligo d(T)15. Recovering and cloning cDNA between 100bp to 750bp.The positive clones were picked out by random sequencing,and then the suspected sequences of ABP genes were obtained.So that, design specific downstream primer on the basis of them.In this case,PCR amplification, cloning,and sequencing using the specific upstream and downstream primers gained the ABP genes of Rana dybowskii skin.Finally using the corresponding specific primers have contrast detection the experimental group and the control group,in order to measure the mRNA expression change trend of ABP genes by RT-PCR.The results showed that the method of extracting total RNA from the skin of Rana dybowskii is a simple and effective method.This experimental system is useful to differentiate samples of which the template amount difference is more than(or equal to) five times.In addition,four novel cDNAs(Brevinin-1ST,Temperin-ST,Dybowskin-1ST and Dybowskin-2ST) have been obtained,which Brevinin-lST,Temperin-ST and Dybowskin-2ST were upregulated and Dybowskin-1 ST did not change significantly under the bacteria stimulating.The up-regulated scope of Brevinin-lST under S.aureus intimidating is higher than E.coli and the change of Temperin-ST is just contrary and the up-regulated scope of Dybowskin-2ST is basically identical under two bacteria.The resoult of this study will provide a good foundation for recombination expression and related study.
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