Identification And Analysis Of The Gene Gcm In Silkworm,Bombyx Mori

Glial cells missing is the earliest Bmdley first reported in 1995 in the change process of neurons and Glial cells play a regulatory role.In fruit flies,GCM encoding a in almost all of the peripheral nervous system and the instantaneous expression of nucleoprotein,both in the central nervous system.In the mutant of GCM disfunction,almost all of the Glial cells cannot,conerted to neurons and outer peripheral nervous system;Expression in GCM,many expected growth for neuron cells into glial cells.GCM for the fate of the glial cells,plays a two-way adjustment role.In the test GCM ectopic expression effect on the development of nerve cells,found the GCM can cause the nerve cells of gene expression and the change of its external form.GCM of macrophages in flies development is also a must.In fruit flies blood progenitor cells to differentiate into plasma cells and crystal cells in the process of GCM to control effects of the development of plasma cells,GCM/gcm2 by inhibiting the activity and stability of the lz to regulate the size of the crystal cell populations.This puts forward new opinions for the research of fruit flies hematopoiesis mechanism.Although the reports on the GCM is not much,but in the process of its development in the drosophila neural development and blood has important research significance.As a result,the GCM genes is very necessary.Silkworm,as the representative of lepidoptera insects,and with the completion of the genome mapping,silkworm gradually become an ideal model of gene function research.Learn through the literature,there are five types of blood cells of mulberry silkworm,but all sorts of functions and regulatory mechanism of blood cells research has not been clear.This paper based on the silkworm genome database,the silkworm Gcm gene cloning and identification,and to analyze its expression.Finally further research,the function of the BmGcm reveals its role in silkworm blood cell regulation.The main results were as follows:1.Identification of the BmGcm family of silkwormWe analyzed silkworm BmGcm family by analyzing the silkworm genome sequence databases,we find BmGcm family has one BmGcm genes.Using RACE technology,we cloned and obtained its complete cDNA sequence.The genes of BmGcm family located on chromosome 4 with a typical Gem domain.Gem family can be grouped into there categories,namely,GCMa,GCMb and GCM.BmGcm genes in silkworm Numbers for BGIBMGA006182(BmGcm)belongs to the family of GCM.2.The expression analysis of silkworm BmGcm familyPCR was used to detect BmGcm gene expression in embryonic period,which showed that BmGcm gene mainly expressed in embry 3 and 4 days,We analyzed the microarray data and expression pattern of various organizations of 5th instar 3th day show that BmGcm are highly expressed in ovary and testis,and no expression in the blood.Based on microarray data and expression pattern of various organizations of 5th instar 3th day,the age of five three days each organization to semi-quantitative and quantitative analysis,and the chip according to the result of basic consistent.Cluster analysis,according to the results of BmGcm and purple sea urchin and long kiss damselfish close genetic relationship.In sea urchins to GCM study shows that the gene regulating the nervous system in the process of embryo development.But by reading the literature found that the gene can not only control the growth of nerve cells in the drosophila,regulation is of blood cells3.Preparation of BmGcm antibodyThe ORF length of BmGcm is 1341bp,encoding 446aa,predicted isoelectric point of the protein is 5.82,and molecular weight is 50.7KDa.Trough silkworm database,we select BmGcm specific fragment which can encode 446aa,prokaryotic expression,iducing conditions,a large number of induction,and purification of proteins.Antiserum was prepared by immunizing mice.eventually won the BmGcm polyclonal antibodies in mice.ELISA to detect the titer of 1:20 000,Suggests that the immunofluorescence antibody preparation effectively,and western detection,prove that the preparation of antibody is available.Western blotting detected blood cell,the molecular of Bmlozenge is 50KDa;consistent with BmlozGcm prediction of protein molecular weight.Suggests that the immunofluorescence antibody preparation effectively,and western detection,prove that the preparation of antibody is available.4.BmGcm overexpressed in BME-SWU3 line and silkwormWe Successful constructed BmGcm expression vector.And transfected it into BME-SWU3 cell line,the results showed that the gene location to the nucleus.In order to further study the role of the gene,at a cellular level,after expressing BmGcm with Brdu detection,found that after the gene expression suppresses the BME-SWU3 cell proliferation.Further by flow cytometry instrument analysis,studies the genes,cell proliferation is in that period,it was found that the gene inhibition of cell proliferation cells mainly in s phase.After through the silkworm expression of the gene,expression of the gene have been found,the silkworm hematopoietic cells from granular cells to differentiate into plasma cells.