Evolution And Immune Function Of PIP5K Gene In Amphioxus

PI(4,5)P2 is a critical membrane lipid that regulates cell physiological events at the cell surface.PI(4,5)P2 is produced from PI(4)P by type I PI(4)P 5-kinase(PIP5K).PIP2 plays an important regulatory role in the signal transmission and signal transduction process of cell plasma membrane.Recent studies have shown that PIP5 K in mammals can participate in TLR-mediated innate immune regulation by regulating the production of PIP2,which has not been reported in other lower species.Amphioxus,as cephalchord animal,plays a special role in the study of the origin and evolution of animal immune system.Therefore,we studied the immune function and evolution of the PIP5 K gene of Branchiostoma belcheri tsingtaunese using molecular biology and bioinformatics methods.The detail results as following:(1)RT-PCR,RACE and End to End methods were used to obtain the full length sequence of PIP5 K gene of Branchiostoma belcheri tsingtaunese,which was named as Amphi PIP5 K.The full length of Amphi PIP5 K gene is 2473 bp,with a 3 'UTR fragment length of 154 bp and a 5' UTR fragment length of 249 bp,including 2,070 bp ORF region encoding 689 amino acids.Amphi PIP5K-flag expression vector was constructed by DNA recombinant technology.The 75 k Da specific protein was successfully expressed through eukaryotic expression experiments.The specific protein was further verified as recombinant Amphi PIP5 K protein by Western blot.(2)To explore the origin and evolution of PIP5 K family genes: first of all,the PIP5 K family genes in different evolutionary status of species were analyzed by homologous search,the results showed that only one member of the family of PIP5 K was found in invertebrates and jawed vertebrates(we are named it PIP5K).While three members of PIP5 K family(PIP5K??PIP5K??PIP5K?)were found in the jawed and higher species.PIP5KL1 as a new member of the family of PIP5 K,was found from echinoderms to mammals,lost in birds;Second,the sequence alignment and structure domain analysis showed Amphi PIP5 K gene contains a conservative PIPc domain,phylogenetic analysis showed the Amphi PIP5 K gene and PIP5 K from invertebrates gather together,and PIP5K??PIP5K??PIP5K? of vertebrates clustered together.The genome structure and synteny analysis showed that the amphioxus Amphi PIP5 K gene should be the orthologous to PIP5K? genes.(3)In order to analyze the immune function of Amphi PIP5 K gene,the temporal and spatial expression patterns of Amphi PIP5 K gene were analyzed by real-time quantitative PCR.The expression of Amphi PIP5 K gene was up-regulated at both 4h and 10 h after LPS stimulation.These results indicated that Amphi PIP5 K gene involved in innate immune response in Branchiostoma belcheri tsingtaunese.(4)miRNA play an important role in animal immune regulation.In order to analyze the regulation effect of miRNAs on Amphi PIP5 K gene,we screened bbe-miR-2013 which may target Amphi PIP5 K gene using miRanda and RNA22 software,and further verified that bbe-miR-2013 can inhibit the expression of Amphi PIP5 K gene by targeting the 3'UTR region of Amphi PIP5 K using the Dual-Luciferase Reporter Assay System.In this study,Amphi PIP5 K gene was cloned from Branchiostoma belcheri tsingtaunese,the function and evolution of PIP5 K gene in amphioxus were analyzed,which provided the insight for the study of the innate immunity in amphioxus.