Molecular Evolution Of Lamprey Pdcd Gene Family And Study Of Apoptosis Mediated By L-PDCD5

Programmed cell death(PDCD)family plays a significant role in the regulation of cell survival and apoptosis.In order to further understand the evolution and molecular functions of pdcd gene family,we screened three generation sequencing library and the primary analysis of ESTs(Expressed Sequence tags)from Lampetra morii Berg.For the first time,we found a gene family similar to programmed cell death in lamprey,which provides a basis for the study of the structure and function of PDCD in the evolutionary process.The research analysis indicated that the nucleotide and deduced amino acid sequences of lamprey pdcd gene family share high sequence similarity with human pdcd gene family.Based on the above observations,we named these genes L-pdcd2,L-pdcd4,L-pdcd5,L-pdcd6 and L-pdca10.In our study,we analyzed the orign of PDCD family from vertebrates,including direct homologues,conserved homologies,functional domains and phylogenetic trees.It was found that there were different domains and tertiary structures among the L-PDCD protein family,but there was no significant difference with other species.In the evolution of pdcd gene family,lamprey were highly conserved with other species.In addition,genetic maps between jaw vertebrates and jawless vertebrates were compared to examine the homologous relationship of L-pdcd gene family.To study the distribution of L-pdcd gene family and the immune response to pathogens,there were significant differences in the distribution of L-pdcd gene family members in different tissues of lamprey by semi-quantitative analysis,and they all showed broad-spectrum expression.The results of quantitative real-time PCR(Q-PCR)at transcriptional level revealed that the L-pacd gene family responded to the stimulation of gram-negative/positive bacteria and viruses by inhibiting or interfering with cell apoptosis.In order to detect changes of L-pdcd family protein levels stimulated by pathogens,Western Blotting experiment and liquid chromatography mass spectrometry(LC-MS)proved that L-PDCD5 itself was stable in the form of trimer in lamprey.When lampreys were stimulated by different pathogens,L-PDCD5 participated in immune response in the form of dimer or monomer.In the study of protein function,result of confocal and flow cytometry showed that rL-PDCD5 protein could enter the nucleus by endocytosis to induce apoptosis after adding rL-PDCD5 protein into the leucocytes of lamprey and human MCF-7 cells.In addition,these genes were overexpressed in cells to study their role in apoptosis.Laser confocal detection showed that the localization of L-PDCD2/5 protein was different in HeLa cells.With the prolongation of L-pdcd5 gene transfection time in HeLa cells,some phenomena such as membrane bubbles,changes in nuclear morphology and granular appearance of L-PDCD5 protein were observed in living cell workstations.CCK8 and flow cytometry experiments showed that overexpression of L-pdcd2/5 genes could induce cell apoptosis,but overexpression of L-pdcd5 gene could induce cell apoptosis more effectively.In order to study the mechanism of L-PDCD5 activating apoptosis regulation,real-time quantitative PCR and Western Blotting results found that the cell changes caused by overexpression of L-pdcd5 were related to BAX(BCL-2 Associated X Protein)and TP53(Tumor suppressor gene)signal transduction pathway.In conclusion,the bioinformatics data in this paper can not only better explain the evolution of L-PDCD family,but also reveal the conservativeness of L-PDCD family in apoptosis process,and provide a new perspective for the study of immune regulation mediated by PDCD family in lamprey.In addition,the immunological function of L-PDCD5 in L-pdcd gene family members was further explored in this paper,which proved that L-PDCD5 could play an important role in the cellular immune response and immune defense of lampreys.