Molecular Cloning And Expression Analysis Of Three Antimicrobial Peptides(LEAP-2??-defensin?BPI) Genes In The Japanese Eel(Anguilla Japonica)

Antimicrobial peptides(AMP)are diverse group of biologically active molecules with antimicrobial properties,playing important roles in host innate immune response,and having a bright prospect in theory researching and application.In this study,a total of five antimicrobial peptide genes(grouped into three family)have been cloned in Japanese eel(Anguilla japonica).Their expression profiles in response to LPS,Poly I:C and Edwardsiella tarda challenged were also investigated.Our results may being,at least to some extent,significantly important in understanding the antibacterial mechanism of the innate immune system in Japanese eel.1?Liver-Expressed Antimicrobial Peptide-2: In the present study,a LEAP-2 gene(AjLEAP-2)was cloned from Japanese eel by RACE PCR.The mature peptide contains a characteristic motif C-X5-C-X4-C-X4-C which is common in other vertebrate LEAP-2.Analysis of AjLEAP-2 genomic organization revealed three exons separated by two introns,which was similar to that in other vertebrates.The AjLEAP-2 gene was transcriptionally expressed in all the detected tissues/organs from Japanese eel,and the highest mRNA expression was observed in liver,followed by intestine.However,the mRNA expression level of AjLEAP-2 in it was only 1/130 of that in liver.Additionally,AjLEAP-2 was highly transcriptionally expressed in glass eel too,which was almost twice the amount in elver intestine.Upon intraperitoneal injection with LPS and E.tarda,the expression of AjLEAP-2 was significantly boosted in blood and reached the peak at 16 h post challenge,with 86 folds and 12 folds higher than the control group respectively.Moreover,a significantly up-regulated transcription expression of AjLEAP-2 in intestine was respectively found at 72 h and at 8 h post challenged with LPS and E.tarda,respectively.Meanwhile,the transcript level of AjLEAP-2 in blood was significantly down-regulated at 24 h after challenged with Poly I:C.These results suggested that AjLEAP-2 may play an important role in innate immunity of Japanese eel against bacterial infection.2??-defensins: Two defensin genes(Ajdefensin1 and Ajdefensin2)were cloned from Japanese eel by RACE PCR.They shared a low amino acid identity(~25.3%)to each other,but both contained a characteristic motif-C-X5-C-X3-C-X11-C-X5-CC-,which was also seen in other teleost and vertebrate counterparts.Both the Ajdefensin genes had a similar three-exon genomic organization,which was also conserved in vertebrates.However,the two defensin showed a distinct mRNA expression profile.In elver,the highest mRNA expression of Ajdefensin1 was observed in liver,while that of Ajdefensin2 was in fish blubber.Additionally,the two Ajdefensin genes were highly expressed in glass eel too.The amount of Ajdefensin1 mRNA was almost twice as much as Ajdefensin2,and is just lower than the liver.Upon PolyI:C stimulation,the expression of Ajdefensin1 was significantly increased in intestine and stomach,with 5 folds higher than the control group at 8 h and 16 h respectively,but significantly down-regulated in kidney at 24 h.Upon E.tarda challenge,significantly up-regulated expression(about 5 folds)of Ajdefensin1 in intestine was found at 8 h.Moreover,significantly increasing expression of Ajdefensin1 in intestine was found at 8 h and at 16 h respectively in response to LPS stimulation,but down-regulated expression in stomach.The expression of Ajdefensin2 was significantly increased in skin after stimulated with PolyI:C.Our results suggested that Ajdefensins may play an important role in innate immunity of Japanese eel against bacterial and virus infection.3?Bactericidal/permeability-increasing protein: Two BPI genes(AjBPI1 and AjBPI2)were cloned from Japanese eel by RACE PCR in the present study.Their identity is 25.3%.AjBPI2 has a signal peptide with 19 amino acids,but AjBPI1 does not have.Both of them contain conserved domains of BPI in its teleost and vertebrate counterparts.The domains from the N-terminus to C-terminus in order are: LPS binding domain,one pair of disulfide bonds,proline-rich central domain.The AjBPIs gene was expressed in all the detected tissues/organs from Japanese eel elver,and they have the similar expression profiles,They express high levels in organs which it contains a high amount of blood.AjBPI1 expression in descending order elver's tissue/organ: liver,middle kidney,spleen,head kidney,skin,blood,intestine,gill,heart,stomach,fish blubber,sexual gland.The expression of AjBPI2 in middle kidney,head kidney and bloood is far higher than other tissue/organ.Additionally,AjBPIs was lowly expressed in glass eel.Upon intraperitoneal injection with LPS,the expression of AjBPI1 was significantly boosted in middle kidney,but significantly reduced in head kidney.The expression of AjBPI2 was significantly up-regulated in gill and middle kidney.Poly I: C can stimulation the expression of AjBPI1 significantly increased in middle kidney and spleen,however AjBPI2 significantly increased in blood,gill,spleen and middle kidney.The results showed of peripheral blood leukocytes in vitro,upon intraperitoneal injection with Poly I: C,the expression of AjBPI1 was boosted expression nearly 200 folds,AjBPI1 was aslo boosted expression about 20 folds.These results suggested that AjBPIs may play an important role in innate immunity of Japanese eel against bacterial and virus infection.