Study Of Molecular Evolution Of Phospholipase A₂ Group IIA In Rats

Objective:To study the characteristics of molecular evolution of rat's phospholipase A2(PLA2) group IIA and to understand the cause of molecular structure to form , which can provide information for synthesis and reconstruction of PLA2 molecule.Methods:All known cDNAs and genes data of rat's PLA2 group IIA were collected and their CDS of DNA and sequenses of amino acid were compared,to analyze base substitutions of synonymous and nonsynonymous,transition and transversion,and the change of substitutions of amino acid's position and their feature,etc.Results:1.The numbers of nonsynonymous substitutions of the PLA2 group IIA gene were higher than synonymous substitutions , and the transversions were higher than the transitions; the substitutions of nucleotide were mostly located on the second positon of codon,the others were located on the first and the third positon; 2.The replacement of nucleotides and amino acids mostly located on the C-terminal part of the protein; 3.Some substitutions of amino acid changed their character.Conclusions:1.Acclerated evolution occurs in the PLA2 group IIA gene of rat.It may be associated with concerted evolution; 2.The basic structure and function of rat's PLA2 group IIA may be related to the N-terminal part; 3.The character change of some amino acids may influence function and structure of PLA2 group IIA molecule. Objective:To study the properties of PLA2 group IB and group IIA'mRNA nucleotide substitutions and amino acid substitutions from different rats.To understand the characteristics of molecular evolution of rat's PLA2 group IIA.Methods: 21 rats were prepared and total RNA was extracted by trizol from the pancreatic gland and the lung that each weighted 50mg～100mg,RT-PCR was performed to amplify the cDNA of PLA2 group IB and group IIA,the products were conducted by electrophoresis;Then the products of RT- PCR were sequenced to compare their sequences,including substitutions of nucleotide and amino acid , sequencing map , then to analyze theircharacteristics of molecular evolution. Results:1.Among the 21 rats,it was found that PLA2-IIA group had 2 SNPs,but PLA2-IB group had only 1 SNP;PLA2-IIA group had 9 times mutations,PLA2-IB group had 2 times mutations; 2.There were heterozygosis and homozygosis at SNP loci chromatography; 3.Total 11 times mutations concentrated on 5 rats.Conclusions:1. PLA2 group IIA gene evolves faster than PLA2 group IB gene. It`s a phenomenon of mosaic evolution of molecular level; 2.There are heterozygotes and homozygotes on the sequences of PLA2 group IIA and group IB. Objective:1.To explore the influence of high glucose(25mM) and low glucose(5.55mM) medium to phospholipase A2(PLA2) group IIA's mRNA transcription of rat liver stellate cell; 2. to underatand whether or not that the gene PLA2 group IIA cultured in vitro have more mutations.Methods:1.Rat liver stellate cell line were cultured under two kinds of DMEM medium,one was high concentration of glucose(25mM),the other was low(5.55mM).Then PLA2 group IIA's mRNA transcriptional level was compared by RT-PCR,electrophoresis and followed by semi-quantity analysis; 2.RT-PCR was performed to amplify the cDNA of gene PLA2 group IIA when the rat liver stellate cell line were passaged about 20 times.The products of PCR were sequenced and then were compared with the sequence of rat gene PLA2 group IIA which were obtained from the last study to detect its mutations.Results:1.There weren't remarkable diference between the electro- phoresis images which were obtained by method of RT-PCR from the rat liver stellate cell line cultured by two kinds of concentration glucose medium;2.There was not mutation in PLA2 gene in cultured cell line.Conclusions:1.The transcription of gene PLA2 group IIA'mRNA of rat liver stellate cell can't be influenced under different concetration of glucose; 2.It suggests that rat liver stellate cell is stable when they are cultured in vitro and are passaged about 20 times. Phospholipase A2 is an enzyme which can catalyze hydrolysis of ester bond of phospholipids,leading to the production of free fatty acids and lysophospholipids and causing inflammatory disorders.If the effect of phospholipase A2 is inhibited , many inflammatory disorders may be treated.Many phospholipase A2 inhibitors have been found and a few inhibitors have been used in clinical trials , which have made many valuable achievements.PLA2 inhibitors are expected as a new type of drug.