Cloning,expression And Evolutionary Analysis Of Monoamine Oxidase Gene From Branchiostoma Tsingtauense

Monoamine oxidase is a flavin protein that exists on the mitochondrial membrane in the form of dimer,which catalyzes the oxidative deamination of monoamines.According to its sensitivity to substrates and inhibitors,monoamine oxidase can be divided into two types:MAO-A and MAO-B,which are likely to evolve from a same ancestor.In this paper,we will introduce the research progress of MAO on the classification and distribution,gene,kinetic mechanism,chemical mechanism,function and inhibitor,and discuss its future research direction.Amphioxus,as a transitional species between invertebrates and vertebrates,has become an important model animal for studying the origin and evolution of vertebrates.It plays a key role and made an important contribution in the field of life science,such as cytogenetics,developmental biology,comparative immunology and so on.1.In this paper,the complete cDNA sequence of monoamine oxidase gene from Branchiostoma tsingtauense was cloned by PCR amplification and RACE for the first time.Its cDNA sequence consists of 2170 bp,including 148bp of 5’UTR,420bp of3’UTR,1602 bp of ORF.The start codon ATG and the stop codon TAA are located at position 149-151 and 1748-1750,respectively.2.The gene of monoamine oxidase from Branchiostoma tsingtauense was analyzed by bioinformatics technique,which encods a deduced protein of 533 amino acids with a molecular weight of 58383.54Da and a p I of 8.19.It was predicted that the protein had a transmembrane structure,which is located in the C-terminal region,and generally belonged to the amine oxidase family.The protein contains one flavinamine oxidoreductase domain,one lysine-specific histone demethylase domain,one monoamine oxidase domain.The phylogenetic tree shows that the evolutionary position of monoamine oxidase gene from Branchiostoma tsingtauense lies between the two big branchs of vertebrates and invertebrates,and has close relatives with invertebrates.3.The prokaryotic expression vector of MAO-pET-28b（+）was constructed by gene recombination technique,and the target protein was expressed after IPTG induction.The protein was preliminarily identified by Westernblot.The results showed that the target protein was derived from the monoamine oxidase gene of Branchiostoma tsingtauense.4.The tissue expression pattern of monoamine oxidase gene from Branchiostoma tsingtauense was studied by in situ hybridization and fluorescence quantitative PCR.The results of in situ hybridization showed that the expression of MAO was observed in the epidermis,notochord and peribranchial cavity epithelium of amphioxus,and its activity was the highest in the notochord,indicating that the notochord may be involved in regulating directional movement and inducing cell differentiation of amphioxus.Fluorescence quantitative PCR was used to analyze the effects of Cu²⁺,Staphylococcus aureus and Vibrio parahaemolyticus.The results demonstrated that concentration of Cu²⁺was positively correlated with the expression of MAO gene,and the relative expression reached the peak in Branchiostoma tsingtauense when the concentration of Cu²⁺was 0.05mg/L.Compared with control group（PBS）,there was significant difference gene expression after stimulation of Staphylococcus aureus or Vibrio parahaemolyticus.The relative expression of MAO gene increased at 4h stimulated by Staphylococcus aureus and 2h,6h and 10h stimulated by Vibrio parahaemolyticus.The relative expression level was the highest at 4h after Staphylococcus aureus challenge,and at 6h after Vibrio parahaemolyticus challenge,which imply that the speed and degree of reaction that MAO participates in the regulation are different under the influence of different bacteria.The above experimental results proved that MAO gene could be related to the immune function of Branchiostoma tsingtauense.