Cloning,Structure,Expression And Evolution Of Cathelicidin-AL And Cathelicidin-RC2 From Anurans

Approximately 3.5 hundred millions years ago,the ancestors of amphibians began to transition from water to land and evolved a series of changes and characteristics adapted to the terrestrial habitat.Thus,the amphibians are special and siginificant groups in the evolutionary history of vertebrate,and they have attracted widely interests of the evolutionarists and ecologists.Due to the complex and changable ecologicial environmentsduring the origin and diversification of the amphibians as well asthe uncovered skin and lack of stratum corneum,immune adaptation was the first question for the amphibians to adapt to the pathogens changes they faced.Previous analyses suggested the acquired immune system evolved not well in amphibians and the innate immune system plays significant roles during their adaptation to the complex habitats.Antibacterial peptides,the key components of the innate immune system,was regarded to have broad spectrum antibacterial activity and plays key roles during their response to the pathogens and the exposure for amphibians.The Cathelicidins are one of the important antibacterial peptide,however,the composition,structure and evolutionary patterns of cathelicidins in amphibians are still unknown to date.In this study,the c DNA sequences of cathelicidin-AL and cathelicidin-RC2 were cloned and sequenced in Rana chensiensis by RACE.The analysis of sequence alignment,protein prediction and phylogeny reconstruction and other bioinformatic analyses were conducted to reveal the amino acids,structure domain,the proteincharacteristics and the evolutionary history.Furthermore,real-time fluorogenic quantitative PCR was used to determine the expression patterns of cathelicidin-AL and cathelicidin-RC2 in R.chensinensis.Finally,a series of primers were designed based on the newly sequences obtained in this study,and the cathelicidin-AL and cathelicidin-RC2 were sequenced in anuran representatives of different habitats.The tree of cathelicidin across vertebrate and amphibians were reconstructed based on BI and ML analysis.The PAML was also used to test the evolutionary patterns of cathelicidin-AL and cathelicidin-RC2 in amphibians.The following are the results of this study:(1)The c DNA sequence of cathelicidin-AL and cathelicidin-RC2 were first cloned and sequenced in R.chensiensis.The length of the c DNA of cathelicidin-ALin R.chensiensisis 677 bp,and the CDS is 561 bp which encodeda protein of 186 amino acids.The c DNA of cathelicidin-RC2 in R.chensiensisis 779 bp in length,and the CDS is 480 bp encoding a protein of 159 amino acids.The site V132 was used as the restriction site in cathelicidin-ALfor R.chensiensis,and the mature peptide is consited with 54 amino acids.The site K123 was found to be the the restriction site in cathelicidin-RC2,and it encoded a mature peptide of 36 amino acid.The length of both CDS of cathelicidin-AL and cathelicidin-RC2 in other anurans differed from species,the lengths of cathelicidin-AL ranged 543-552 bp,and that of cathelicidin-RC2 ranged468-501 bp.Two disulfide bonds were found at C80-C92 and C103-120 in cathelicidin-AL,whereas only one disulfide bond was found in cathelicidin-RC2 although the existence of 4 cysteines at C75-C86 and C97-C108.(2)The tissue distribution and expression analysis in R.chensinensis show that both cathelicidin-AL and cathelicidin-RC2 expressed in lung,skin,stomach,kidney,spleen,brain,and liver,but not in heart.The highest expression of cathelicidin-AL was in liver and followed by brain and spleen,whereas it reached the highest level in brain for cathelicidin-RC2 and followed by liver and spleen.The level of expression of both cathelicidin-AL and cathelicidin-RC2 in lung and skin was low.(3)The phylogenetic renconstructionof representative vertebrates suggested the earlier origin of cathelicidin-AL than cathelicidin-RC2.According to the phylogeny of reprsentative known cathelicidins,the basal postion of cathelicidin-AL of R.chensinensis was revealed,where as the cathelicidin-RC2 have a close relationship with reptiles,birds and mammals and they togther foremed a branch.In addition,representatives of cathelicidin-AL and cathelicidin-RC2 formed a group,respectively,according to the phylogeny of the known amphibian cathelicidins,and it also revealed the similar results that cathelicidin-AL appeared earlier.(4)The selectivepressure analysis suggested free ratio model of cathelicidin-AL/-RC2 is better than one ratio model,and it indicated the slective pressure was different among different lineages.Positively selection was detected along the ancestral branch of the genus Odorrana,Amolops wuyiensis,Pelophylax nigromaculatus,Bufo gargarizans in cathelicidin-AL based on the branch specific model.Significant positively selection was also detected along the branch of O.tormota,R.chensinensis,P.nigromaculatus,the common ancestor of Rhacophoridae and Dicroglossidae in cathelicidin-RC2.Interestingly,the branch site model also detected one positively selected sites(138V)in O.tormota.Due to the variable mature peptide of the end of cathelicidins,and different slective pressure detected along different branches in cathelicidin-AL and cathelicidin-RC2,they might play important roles during the adaptation to different habitats for amphibians.