Study On Function And Regulation Mechanism Of Two Antimicrobial Peptides Genes In Trachinotus Ovatus

Trachinotus ovatus is widely cultured in the Asia-Pacific region due to its rapid growth rate,high economic value and suitability for cage culture.With the development of the aquaculture industry,the amount of antibiotics is gradually increasing,which not only pollutes the environment,but also causes pathogens to develop resistance.The antibacterial mechanism is different between antibacterial peptide and antibiotics.Antibacterial peptide is difficult to produce drug resistance and possess potential substitute for antibiotics.Screening related antibacterial peptide genes can be used for breeding of excellent varieties.To identify the function and regulation mechanism of the antibacterial peptide gene in T.ovatus,the full-length c DNA sequences of LEAP-2 and NK-lysin genes were cloned,the structural features of the genes were analyzed by bioinformatics.The tissue distributions and expressed pattern of LEAP-2 and NK-lysin with Photobacterium damselae stimulation were analyzed by q RT-PCR.Moreover,the mature peptide was cloned and the protein successfully expressed in vitro by constructing the prokaryotic expression vector.The LEAP-2 and NK-lysin gene promoter sequences were amplified,and the core promoter was screened by constructing a luciferase reporter gene recombinant plasmid.Constructing transcription factor binding site mutant by PCR technique and the main transcription factor was screened.The specific research results were as follows.(1)The c DNA sequence and gene sequence of LEAP-2 and NK-lysin were cloned in this study.The full-length LEAP-2 c DNA was 1758 bp,which comprised a 5'-UTR 250 bp,an ORF of 321 bp,a 3'-UTR of 1187 bp,encoding 106 amino acids,which consisted of a N-terminal signal peptide 29 aa,a precursor peptide 31 aa,a mature peptide 46 aa,the mature peptide included 4 cysteines which constituted 2 pairs of disulfide bonds.The full-length LEAP-2 DNA was 3096 bp,which comprised three exons and two introns,the LEAP-2 genes structure of mammals,birds,amphibians,reptiles and teleost fish were identical.The full-length NK-lysin c DNA was 731 bp,which comprised a 5'-UTR 63 bp,an ORF of 444 bp,a 3'-UTR of 224 bp,encoding 147 amino acids which consisted of a conserved saposin B domain and six conserved cysteines which formed three pairs of disulfide bonds.The full-length NK-lysin DNA was 1827 bp,which comprised five exons and four introns,the NK-lysin gene structure of mammals,reptiles,Atlantic salmon and Oryzias latipes were identical.However,four exons and three introns of NK-lysin were in Salvelinus alpinus and Larimichthys crocea..The LEAP-2 and NK-lysin gene of T.ovatus was highest expressed in the liver and gill,respectively.After stimulation with P.damselae,the expression levels of LEAP-2 and NK-lysin were significantly increased in liver,head kidney,intestine and spleen.(2)In this study,the mature peptide of mLEAP-2 and p ET-32a/m LEAP-2 expression vector was cloned and constructed,respectively.The induction conditions were 37°C,0.8 mm IPTG and 220 rmp for 4 hours.However,the protein could not express in E.coli BL21 or rossate,subsequently,the p GEX-6P-1/m NK-lysin expression vector was constructed and expressed in E.coli BL21.The induction conditions were 18 °C,0.1 mm IPTG and 220 rmp for 6 hours.The antibacterial activity was detected based on inhibition zone method and the microdilution method.The results showed that the m LEAP-2 and m NK-lysin recombinant proteins in golden pompano have antibacterial activity against gram-positive bacteria,gramnegative bacteria and fungi.(3)The luciferase report of five different length deletion fragments(L1,L2,L3,L4,L5)were constructed from promoter region(-1575 bp to +251 bp),,then transfected into 293 T cells.The results showed that the L3(-659 bp to +251bp)was the highest activity and was defined as the core region of LEAP-2 promoter.The seven predicted transcription factor binding sites were deleted by PCR technology,then constructed luciferase report vectors and transfected into 293 T cells.The result showed that the mutation USF transcription factor binding site caused the active significantly decreased.Moreover,the luciferase report of five different length deletion fragments(L1,L2,L3,L4,L5)were constructed from promoter region(-854 bp to +40 bp),then transfected into 293 T cells.The results showed that the N3(-476 bp to +40 bp)was the highest activity and was defined as core region of NK-lysin promoter.The seven predicted transcription factor binding sites were deleted by PCR technology then constructing the luciferase report and transfected into 293 T cells.The result showed that C/EBPalp and Oct-1 transcription factor binding site mutation were significantly active increased.