Identification Of BmCKI And Its Function In The Regulation Of The Endocycle Of Silk Gland Cells

Bombyx mori is an important economic insect which synthesizes the silk proteins.The silk gland is the special silk-producing organ of the silkworm larvae.Efficient endoreplication in silk gland cells is the premise of silk protein synthesis.Endocycle,also known as‘endoreplication'cycles,is a common cell cycle variant in which cells successively duplicate genomic DNA without segregating their chromosomes during mitosis,and generate cells referred to as‘polyploid'.The silk gland morphogenesis is complete post silk gland cells undergoing about ten cycle of mitosis during embryonic phase.Silk gland contains approximately 1080 cells and can be anatomically and physiologically divided into three distinct regions:the anterior silk gland?ASG?,the middle silk gland?MSG?and posterior silk gland?PSG?.All the cell division cycles of this tissue are confined to the embryonic stage and extensive growth of silk glands during the larval stages is due to increase in tissue mass and not cell number.DNA replication in these cells continues without mitosis.MSG and PSG operate for 17¹9rounds of endocycle during the larval stages of development,resulting in an increase in the DNA content by 2¹⁷²¹⁹ times?about 400,000 times the haploid genomic content per nuclei?.This allows an increase in the gene copy number per unit cell,which is beneficial for the rapid synthesis of silk proteins.The study of the endocycle in fruit flies and mice previously,suggested that endocycles use much of the same machinery that regulates mitotic cell cycles.The oscillation of cyclin-CDK activity is also essential for progression through multiple endocycles.The activities of cyclin-CDK complexes are regulated by cyclin-dependent kinase inhibitors?CKIs?.CKIs can bind to cyclin-CDK complexes and inhibit CDKs activities.Compare to endocycles in other organism,the time span of endocycle in silk gland is longer and the efficiency of DNA replication is higher.Elucidation of the endocycle mechanism of silk gland cells will provide important theoretical basis for the genetic improvement of silk glands and increases our knowledge of cell cycle regulation.This study on the identification of cyclin-dependent kinase inhibitor in Bombyx mori?BmCKI?,and explore the mechanism of this gene regulating endocycle of silk gland,which provides a good basis for comprehensive analysing the mechanisms of endocycle of silk gland.The main results and conclusions are as follows:1.Identification and Characterization analysis of BmCKIWe cloned and identified one homogenous gene of the Cip/Kip family in the silkworm and named this gene BmCKI.We obtained two transcripts of the BmCKI gene,and named these BmCKI-L and BmCKI-S.The longer transcript BmCKI-L encodes a protein with 217 amino acids.In contrast to BmCKI-L,the transcript BmCKI-S differs in the 5'UTR,lacks an in-frame portion of the 5'coding region,and initiates translation at a downstream start codon.The BmCKI-S has a shorter N-terminus and encodes a protein with 192 amino acids.Multiple sequence alignments of the Cip/Kip family homologs suggested that both BmCKI-L and BmCKI-S had high amino acid identity with other homologs in their conserved domains,which contain a cyclin-binding domain and CDK-binding domain.Analysis of the phylogenetic tree showed that BmCKI-L,BmCKI-S and Drosophila melanogaster dacapo form one group.These results indicate that BmCKI is a homolog of the Cip/Kip family.Subcellular localization of BmCKI-L and BmCKI-S was detected by immunofluorescence.The results indicated that BmCKI-L and BmCKI-S were localized in the nuclei.To determine which amino acids are essential for the nuclear localization of BmCKI,we constructed a series of truncated mutants of BmCKI-L by fusing the N-terminus of the GFP tag.The subcellular localization of BmCKI-L mutants was assessed by immunofluorescence after transient transfection in BmN-SWU1 cells.The results suggest that the amino acid residues 181-210 contain the core NLS domain and contribute to the nuclear localization of BmCKI.This region contains an unconventional putative bipartite NLS with a 16 amino acid linker.The linker NLS of BmCKI is enriched in histidine residues which can also make the NLS positively charged.The qRT-PCR analysis of the relative expression level of BmCKI-L or BmCKI-S in different silkworm tissues suggested that BmCKI-L and BmCKI-S have different expression patterns in silkworm tissues,indicating that they may play different roles in different tissues.2.BmCKI is involved in regulating the silkworm cell cycleTo investigate whether BmCKI can regulate cell cycle progression,BmCKI-L and BmCKI-S were overexpressed in BmN-SWU1 cells.Flow cytometry analysis was performed to determine its cell cycle distribution according to DNA content.Compared to the control group,the proportion of G1 phase cells in BmCKI?both BmCKI-L and BmCKI-S?overexpression group were significant higher;the proportion of cells in the S phase and the G2/M phase were both lower.This indicated that overexpression of BmCKI led to cell cycle arrest in the G1 phase.To further determine the effect of DNA replication after BmCKI overexpression,the BmCKI transfected BmN-SWU1 cells were incorporated with 5-bromodeoxyuridine?BrdU?.BrdU-labeling drastically decreased in cells overexpressing BmCKI compared with the control,indicating that the relative rate of DNA synthesis after BmCKI overexpression was greatly reduced compared to controls.We conclude that overexpression of BmCKI arrested the cell cycle in the G1phase and inhibited DNA replication so that cell was blocked to enter into the S phase.Meanwhile,the proliferation of BmN-SWU1 cells was analyzed by using the CCK-8assay.The results showed that overexpression of BmCKI-L or BmCKI-S inhibited the proliferation of the BmN-SWU1 cells.To further verify that BmCKI is involved in cell cycle regulation,microRNA-mediated RNAi was performed to silence the expression of BmCKI in BmN-SWU1 cells.Flow cytometry analysis was performed to determine its cell cycle distribution according to DNA content.Compare to the control group,the proportion of G2/M cells in BmCKI overexpression group was significant higher;and the proportion of cells in the G1 phase and the S phases were both lower.These results showed that knockdown of BmCKI led to cell accumulation at the G2/M phase.To further determine the effect of DNA replication after BmCKI knockdown,the BmCKI transfected BmN-SWU1 cells were incorporated with BrdU.BrdU-labeling drastically increased in cells knockdown of BmCKI compared with the control,indicating that the relative rate of DNA synthesis after BmCKI knockdown was greatly increased compared to controls.Meanwhile,the proliferation of BmN-SWU1 cells was analyzed by using the CCK-8assay.The results showed that knockdown of BmCKI-L or BmCKI-S promoted the proliferation of the BmN-SWU1 cells.3.BmCKI is involved in regulating the growth and development of silkworm.To confirm the function of BmCKI in individual,we constructed transgenic silkworm with overexpressing of BmCKI-L,and named BmCKI-L-OE.Observing the embryos of the BmCKI-L-OE strain G1 generation,we found that about 2/3 embryos were lethal and about 1/3 embryos could be hatched normally.The larvae of BmCKI-L-OE strain exhibited developmental retardation to different degrees and died gradually.BmCKI-L-OE larvae were smaller than Dazao of the same instar.The survival rate of BmCKI-L-OE and Dazao strains indicated that BmCKI-L-OE strain possessed embryonic lethal,larva developmental retardation and lethal.Meanwhile,the anatomy of BmCKI-L-OE and Dazao silkworm showed that multiple tissues of BmCKI-L-OE strain were abnormally developed.For instance,the malpighian tubule of BmCKI-L-OE was atrophic and yellower than that of Dazao;branches of the tracheal in BmCKI-L-OE silkworm were reduced,and the length of the tracheal was shortened;the silk gland of BmCKI-L-OE was significant smaller than that of Dazao;the fat body hardly existed in BmCKI-L-OE strain.These results suggested that BmCKI-L might regulate the growth and development of silkworm.4.BmCKI is involved in regulating the endocycle of silkworm.In order to explore the role of BmCKI in the endocycle of silk gland,we constructed transgenic silkworm with overexpressing of BmCKI-L in silk gland by using silk gland specific promoter ser1,and named Ser1^P-BmCKI-L-OE.Observing the phenotypes of Ser1^P-BmCKI-L-OE,statistical analysis showed that Ser1^P-BmCKI-L-OE exhibited size reduced and weight loss than normal Dazao.Observing the phenotypes of Ser1^P-BmCKI-L-OE silk gland,the results showed that the silk gland of Ser1^P-BmCKI-L-OE silkworm exhibited size,length,diameter and weight of silk gland reduced than normal Dazao.These results suggested that overexpression of BmCKI-L in silk gland inhibited the development of the silk gland of silkworm.Economic character analysis of Ser1^P-BmCKI-L-OE transgenic strain showed that the weight of cocoon,pupa,and cocoon shell,and the rate of cocoon shell reduced than normal Dazao.These results suggested that overexpression of BmCKI in silk gland reduced the economic character of silkworms.Using frozen sections and DAPI staining detect the DNA content in silk gland of Ser1^P-BmCKI-L-OE transgenic strain.The result showed that DAPI staining in ASG,MSG,and PSG of Ser1^P-BmCKI-L-OE was less than Dazao,suggested that the DNA content of silk gland of Ser1^P-BmCKI-L-OE less than Dazao.BrdU labeling of silk glands in vitro was performed to detect the DNA replication in silk gland of Ser1^P-BmCKI-L-OE transgenic strain.The result showed that BrdU labeling in ASG,MSG,and PSG of Ser1^P-BmCKI-L-OE was less than Dazao,suggested that DNA replication in silk gland of Ser1^P-BmCKI-L-OE less than Dazao.These results suggested that overexpression of BmCKI-L in silk gland inhibited the endocycle progression of silk gland cells.5.Identification of interaction proteins and regulation mechanism analysis of BmCKIUsing yeast two-hybrid technique and immunoprecipitation screened and identified three proteins,BmPCNA,BmRACK1 and BmSTMN interact with BmCKI-L.Proliferating cell nuclear antigen?PCNA?is a processivity factor for DNA polymerase?in eukaryotic cells and is essential for DNA replication and DNA repair.RACK1 is a protein with seven tryptophan-aspartic acid repeats.RACK1 are combined with various proteins in different areas of the cell,so that plays an important role in many basic physiological processes.STMN is a highly conserved protein that is crucial for the regulation of the cell cytoskeleton.The qRT-PCR date showed that BmPCNA had the specific and high-level expression in silk gland,BmSTMN had the specific and low-level expression in silk gland,and RACK1 had the high-level expression in every tissue.Flow cytometry analysis was performed to determine the effects on the cell cycle after overexpression or knockdown of BmPNCA.The results showed that the proportion of G1 phase cells in BmPCNA overexpression group was significant higher compared to the control group;whereas,the proportion of G1 phase cells in BmPCNA knockdown group were significant lower compared to the control group.Meanwhile,the qRT-PCR date suggested that BmCKI-L was upstream of BmPCNA and inhibited BmPCNA.It could infer that BmCKI-L might regulate the cell cycle through binding to and inhibiting BmPCNA.The results of flow cytometry analysis showed that the proportion of G2/M phase cells in BmRACK1 overexpression group was significant higher,and the proportion of G1 phase cells in BmRACK1 overexpression group was significant lower compared to the control group;whereas,the proportion of G1 phase cells in BmRACK1knockdown group was significant higher,and the proportion of G2/M phase cells in BmRACK1 overexpression group was significant lower compared to the control group.Meanwhile,the qRT-PCR date suggested that BmRACK1 was upstream of BmCKI-L and promoted the expression of BmCKI-L.It could infer that BmRACK1 might regulate the cell cycle through binding to and inhibiting BmCKI-L.The results of flow cytometry analysis showed that the proportion of G2/M phase cells in BmSTMN overexpression group or BmSTMN knockdown group was significant higher compared to the control group.Meanwhile,the qRT-PCR date suggested that BmCKI-L was upstream of BmSTMN and inhibited BmSTMN.It could infer that BmCKI-L might regulate the cell cycle through binding to and inhibiting BmSTMN.To sum up,we constructed a possible network of BmCKI regulating the cell cycle.It inferred that BmCKI might regulate the cell cycle including endocycle in multiple pathway:BmCKI might regulate the cell cycle by binding to and inhibiting cyclin-CDK complex;BmCKI might regulate the cell cycle by regulating DNA replication through binding to and inhibiting BmPCNA;BmCKI might regulate the cell cycle by regulating cytoskeletal dynamics through binding to and inhibiting BmSTMN;in addition,BmRACK1 might regulate the expression of BmCKI.