Study On The Effect Of BmGeminin1 To The Silk Gland Cell Endocycle

The endocycle is a kind of cell cycle which widely distributing in the organisms only for DN A replication but not carrying out cell division,and performs different functions in different organisms.The unique cell cycle of the endocycle is widely concerned,studies found that the endocycle in plants is widely involved in the formation and development of germs,roots and fruits.The mammalian endocycle is widely involved in the biological processes such as cell injury repair,tissue growth and development.The silkworm glands have typical nuclear cycle characteristics.Following the limited number of mitosis in the embryonic period,the silk glands cell begin to duplicate DN A in endocycle,and the cells are repeatedly replicated,and the cell volume increases,and then the protein is synthesized.The aim of this study is to provide a clue for the study of the mechanism of cycling in the endocycle of the organism,which is the basis for the analysis of the mechanism of efficient synthesis of silk protein by silkworm gland.This laboratory has previously identified two Geminins,named as BmGeminin1(BmGem1)and BmGeminin2(BmGem2).Studies showed that DNA in cell would happen to repeat replication and to be aneuploidy when BmGem1 was interfered,while DNA content wouldn't change and cell was suspended in S phase when it was overexpressed.In order to explore the role of BmGeminin1 in the endocycle of silkworm silk gland and to analyze its mechanism of regulation of the internal cycle of silk gland,in this study,BmGem1 overexpression and interfering transgenic vector were constructed to acquire the silkworm transgenic material,and the phenotypic characteristics of transgenic lines were observed.And the changes of DNA content and DNA repeat replication of transgenic lines were analyzed.And with the transcriptomics among the transgenic stains,we search the probable key genes involved in regulation of silk gland cell endocycle.The main findings are as follows:1.Structure and identification of BmGeminin1 transgenic lines.Using silk gland-specific promoters Ser1 Promoter and Ser1 p A to construct BmGem1 interference and overexpression transgenic vectors,BmGem1-RNAi and BmGem1-OE transgenic lines,which are specifically interfered with and overexpressed BmGem1,were acquired by microinjection and other transgenic techniques.Further detections,reverse PCR and qRT-PCR,showed that the single copy of BmGem1-RNAi fragment was inserted into nscaf1898 on 13 th chromosome,and it induced BmGem1's significant down-regulation.While the single copy of BmGem1 fragment was inserted into chromosome nscaf2204 on the 19 th chromosome,and it induced BmGem1's significant up-regulation.2.pPhenotype observation and analysis of transgenic strains.Observing the BmGem1-RNAi and BmGem1-OE strains phenotypes,statistical analysis showed that BmGem1-RNAi strain appeared silk body length growth,weight gain phenomenon,the BmGem1-OE strain appeared to shorten the body length of the silkworm,the phenomenon of weight loss.Further analysis of silkworm,statistical analysis of silk gland phenotype,the results showed BmGem1-RNAi silkworm silk glands bigger,while BmGem1-OE lines silkworm silk gland smaller.In order to analyze the phenotypic differences between silkworms and silk glands in the two strains,frozen sections and immunohistochemical DAPI staining were performed on the silk gland tissue.The results showed that the DNA content of silkworm gland cells decreased in the BmGem1-RNAi strains,while DNA content of silkworm gland cells in BmGem1-OE lines increase.The BrdU labeling of silk glands in vitro was further analyzed.The results showed that the DNA repeat replication in BmGem1-RNAi strain was enhanced while the duplication of DNA in BmGem1-OE strain was inhibited.The above results suggest that BmGeminin1 may affect the DN A content of silk gland cells by the regulation of DNA duplication in the nucleus of silkworm gland cells,which may lead to changes in the size of silk gland cells,leading to changes in the size of silk gland,changes in body type in the end.3.Analysis of transcriptome differences among in transgenic strains.In order to analyze the molecular mechanism of BmGeminin1 involved in regulating the nuclear cycle of silkworm gland cells,the transcriptome data of BmGem1-OE,BmGem1-RNAi and Dazao lines were analyzed.26 differences were found in the BmGem1-OE strain compared to Dazao,including 16 up-regulation genes and 10 down-regulation genes;25 different genes were obtained in BmGem1-RNAi strain,all up-regulated.Analysis with KEGG pathway,it showed different expression genes in BmGem1-RNAi mainly involved in Protein processing in endoplasmic reticulum,while BmGem1-OE mainly involved in Oxidative phosphorylation.According to the transcriptome data,three genes which involved in energy metabolism: BGIBMGA006011(NADH dehydrogenase),BGIBMGA006013(flavoprotein)and BGIBMGA009987(NADH dehydrogenase ubiquinone)down-regulated were selected.It is speculated that the excessive accumulation of BmGe mninin1 in the silk gland tissue may cause the electron transport chain to be disordered and affect the normal supply of energy in the DNA synthesis,which leads to the decrease of DNA replication.Meanwhile,two transcription factors were screened: BGIBMGA009027(Sp3)?BGIBMGA008965(bHLH).Sp3 could affect the expression of ?-catenin which was important in Wnt/?-catenin signal pathway,so it was agreed as regulator in silk gland cell endocycle.