Molecular Cloning,gene Expression And Functional Study Of Important Immune-related Genes From Bostrychus Sinensis(Family:Eleotridae)

The Chinese black sleeper?Bostrychus sinensis?,which belongs to the family Eleotridae,Order Perciforms,is a euryhaline fish mainly distributed in intertidal mudflats and has the characteristics of strong vitality,fast growth and cave dwelling.Physiological studies had shown that the Chinese black sleeper has strong resistance to dry dew,and the dry dew does not die for 20 hours,which is very suitable for live transportation.In recent years,Guangxi,Guangdong,Fujian and other coastal areas have set off an upsurge in breeding Chinese black sleeper,making it an important artificial breeding target.With the continuous increase of the breeding area,various breeding diseases including enteritis,dermatosis,vibrio,and parasitic diseases have also appeared one after another,causing great economic losses to the Chinese black sleeper aquaculture.Therefore,better understanding the mechanisms underlying the immune response of B.sinensis to pathogen infection will help us in developing strategies for management of diseases and enhance the healthy development of the mariculture of this commercial fish species.In this study,we isolated a dominant strain from the diseased Chinese black sleeper in Taizhou.The pathogenicity,immunology,and other aspects of the diseased Chinese black sleeper were investigated by means of regression infection,histopathological section,physiological and biochemical and molecular biology.The research would provide reference for the diagnosis and prevention of the disease.Then,we performed transcriptome sequencing and analysis of the spleen after Vibrio parahaemolyticus infection,and screened the following 4 important innate immune genes for analysis.1.molecular cloning,mRNA expression and functional characterization of superoxide dismutase from the Chinese black sleeperThe main function of superoxide dismutase?SOD?is to catalyze O^2-into oxygen?O²?and hydrogen peroxide?H₂O₂?.In this study,two subtypes of the superoxide dismutase gene family of Chinese black sleeper,BsCu/Zn-SOD and BsMn-SOD,were cloned and sequenced.The open reading frame of BsCu/Zn-SOD gen contains 465bp nucleotides and encodes 154amino acids;the open reading frame of BsMn-SOD gen contains 678bp nucleotides and encodes 225 amino acids.The results of sequence analysis showed that BsCu/Zn-SOD has no signal peptide and is an intracellular protein;the Mn-SOD gene contains a 27aa mitochondrial targeting sequence at the N-terminus and is an extracellular protein.BsCu/Zn-SOD and BsMn-SOD genes were expressed ubiquitously in in all examined tissues?liver,spleen,brain,head kidney,muscle,peripheral blood,brain,skin,heart,gill,intestine?,and both of them were abundantly expressed in the muscle.Treatment with Vibrio parahemolyticus?V.parahemolyticus?and Polyinosinic:polycytidylic acid?Poly?I:C??,the expression of BsCu/Zn-SOD and BsMn-SOD mRNA appeared significantly altered in the liver,peripheral blood,head kidney and spleen.2.molecular cloning,mRNA expression and functional characterization of a catalase from the Chinese black sleeperCatalase?CAT?is one of the key enzyme in protect the cell from oxidative damage by reactive oxygen species.In this study,we sequenced and characterized the coding sequence of Bostrychus sinensis catalase gen?BsCAT?in the Chinese black sleeper?Bostrychus sinensis?.The open reading frame of BsCAT is 1575 nucleotides encoding a protein of 525 amino acids,the analyze of amino sequence demonstrated the BsCAT has significantly homology with other species and highly conserved motifs including the proximal active site signature,heme-ligand sequence and three catalytic amino acid His⁷⁵,Asn¹⁵⁸ and Tyr³⁵⁸.Real-time quantitative PCR showed that the CAT gene of Chinese black sleeper was expressed in all examined tissues?liver,spleen,brain,head kidney,muscle,peripheral blood,brain,skin,heart,gill,intestine?,and was abundantly in the liver.Treatment with Vibrio parahemolyticus?V.parahemolyticus?and Poly?I:C?,the expression of BsCAT mRNA appeared significantly altered in the liver,peripheral blood,head kidney and spleen.The purified recombinant BsCAT has optimal activity at 15°C and pH 7.0.3.Molecular cloning,expression analyses and functional characterization of lysozyme?BsLysG and BsLysC?gene from Bostrychus sinensis?family:Eleotridae?Lysozymes are important molecules of the innate immune system and could play vital roles in defense against pathogen infection.Lysozymes,also known as muramidases,catalyze the hydrolysis of?-?1,4?-glycosidic bond between N-acetyl muramic acid?NAM?and N-acetyl glucosamine?NAG?in the peptidoglycan layer of the bacterial walls,leading to lysis of Gram-positive bacterial cell.In this study,two subtypes of the lysozyme gene family of Chinese black sleeper,BsLysG and BsLysC,were cloned and sequenced.The open reading frame of BsLysG gene contains 591bp nucleotides and encodes 196 amino acids,the obtained genome sequence of the BsLysG coding region spans 5046 nucleotides and possesses five exons split by four introns;the open reading frame of BsLysC gene contains 465bp nucleotides and encodes 154 amino acids,the obtained genome sequence of the BsLysC coding region spans 2493 nucleotides and possesses 4 exons split by3 introns.The results of sequence analysis showed that BsLysG has no signal peptide and is an intracellular protein,and significantly homology with other species and highly conserved motifs including three catalytic amino acid His⁷²,Asn⁵⁸ and Asp¹0202 and GLMQ motif(Gly⁹⁷,Leu⁹⁸,Met⁹⁹ and Gln¹⁰⁰);BsLysC was detected that it was a secretory protein with a N-terminal signal peptide,containing two conserved catalytic amino acid,Glu⁵² and Asp⁶⁹,and eight conserved Cys,which form four pairs of disulfide bonds.BsLysG and BsLysC genes were expressed ubiquitously in in all examined tissues?liver,spleen,brain,head kidney,muscle,peripheral blood,brain,skin,heart,gill,intestine?,both of them predominant expression detected in spleen.Treatment with V.parahemolyticusand Poly?I:C?,the expression BsLysG and BsLysC mRNA appeared significantly altered in the liver,peripheral blood,head kidney and spleen.But the higher degree and peak of time of the two genes in different tissues.The purified recombinant BsLysG and BsLysC exhibits optimum enzymatic activity at 35°C?pH 7.0 and 50°C?pH 6.0,respectively.4.molecular cloning,mRNA expression and functional characterization of Hepcidin from the Chinese black sleeperAntimicrobial peptides constitute important components of the innate immune system in many species,including plants,invertebrates,and vertebrates.They play an important role in protecting these organisms against microbial invasion.In this study,we sequenced and characterized the coding sequence of Bostrychus sinensis Hepcidin gene?BsHep?in the Chinese black sleeper.The open reading frame of BsHep is 273 nucleotides encoding a protein of 90 amino acids,the obtained genome sequence of the BsHep coding region spans507 nucleotides and possesses three exons split by two introns.The analyze of amino sequence demonstrated the BsHep has significantly homology with other fishes.The amino acid sequence was consisted of 24aa of signal peptide,40aa of prodomain and 26aa of mature peptide.The mature peptide contains 8 highly conserved Cys,which form four disulfide bonds to maintain the stability of the peptide tertiary structure.Real-time quantitative PCR showed that the BsHep gene of Chinese black sleeper was expressed in all examined tissues?liver,spleen,brain,head kidney,muscle,peripheral blood,brain,skin,heart,gill,intestine?,and was abundantly in the liver,followed by spleen and peripheral blood.Treatment with V.parahemolyticusand Poly?I:C?,the expression BsHep mRNA appeared significantly altered in the liver,peripheral blood,head kidney and spleen.But the higher degree and peak of time of the two genes in different tissues.Hepcidin peptide was synthesized by GenScript Corporation?Nanjing,China?,then utilized the peptide to test its antibacterial activity toward to V.parahemolyticus?E.coli?Staphylococcus aureus and Vibrio harveyi.