Identification Of Gata-binding Protein Family And Functional Analysis Of BmGATA6-like Gene In Silkworm, Bombyx Mori

As an important invertebrate model, silkworm undergoes huge changes during the process of metamorphosis and development, which made it an ideal material for the study of cell proliferation, differentiation, apoptosis, autophagy and other physiological processes. GATA-binding protein family is an important transcription factor family during the development process from nematodes to mammals, and is involved in the regulation of embryonic development, tissue and organ formation and other important life events. In this study, the silkworm GATA-binding protein sequence informations were obtained by using the silkworm genome database. The function of BmGATA6-like gene has been intensively studied and analyzed, which is helpful for the elucidation of molecular mechanism regulating silkworm metamorphosis, and for the comprehensive understanding of insect metamorphosis. That can provide clues for the biological control of pests, even provide reference data for human degenerative diseases. The main results are as follows: 1. Identification of the GATA-binding protein family in silkwormBased on NCBI and the whole genome database of silkworm, five silkworm GATA-binding protein genes were obtained, and they were named BmGATA-A-like, BmGATA-C-like, BmBCF I, BmGATA6-like and BmGATA-X. Similar to those in other species, silkworm GATA-binding proteins also have conserved GATA zinc finger domains. BmGATA-A-like and BmGATA-X contains only one GATA zinc finger domain, but BmGATA-C-like, BmBCF I and BmGATA6-like have two GATA zinc finger domains severally. The microarray data of different tissues on L5D3 silkworm showed that GATA-binding proteins have low expression level in silworm tissues, but there are big differences in expression among members. Evolutionary analysis of conserved domains showed that the members of the GATA-binding protein family of silkworm are mainly associated with the GATA-binding proteins of the insects. Among them, BmGATA-A-like and Pannier of Drosophila were clustered into one class; BmBCF I and GATAa of Tribolium castaneum and Serpent of Drosophila were together as a class; BmGATA6-like and lepidopteran GATA-binding protein gene were clustered into one class; BmGATA-X and Apis mellifera GATA4-like is the most conservative, while Bm GATA-C-like is clustered separately. The multiple sequence alignment between the species in the conserved domain shows that the homology of the GATA zinc finger domain sequences is very high. 2. Cloning and expression analysis of BmGATA6 gene in silkwormBy using the sequence information of the silkworm genome database and the NCBI Blast sequence, the full-length cDNA sequence of BmGATA6 gene was cloned. The BmGATA6 gene full length is 4011 bp, containing 10 exons and 9 introns. BmGATA6 ORF frame is 1974 bp, and encodes 657 amino acids. The predicted protein molecular weight is 70.8kDa, and the isoelectric point is 6.13. The BmGATA6 protein contains two typical GATA zinc finger domains, which are located in the 464 th ~516th amino acid and the 524 th ~576th amino acid, and on line software predicts that it has no signal peptide. The relative expression levels of BmGATA6 gene in the different stages of embryo and different tissues of L5D3 were detected by real-time fluorescence quantitative PCR. BmGATA6 was expressed during the whole developmental stages of embryo, and it was persistent highly expressed in the later stage of embryonic development. The expression of BmGATA6 reached a peak in the 6th day of embryonic development and the hatching larvae respectively. Tissue expression profile of L5D3 showed that BmGATA6 is widely expressed in tissues, and is highly expressed in midgut. The expression of BmGATA6 was detected in midguts and hemocytes at different stages, in which BmGATA6 was expressed highly and lowly respectively. The consequence in midgut showed that BmGATA6 is expressed sostenuto, and has abundant quantity during moulting stage and metamorphosis stage. Similar to that in midgut tissue, the expression of BmGATA6 in hemocytes is significantly high during moulting stage and metamorphosis stage. The localization of BmGATA6 was performed in midgut tissue and hemocytes by immunofluorescence, results showed that BmGATA6 is highly expressed in all types of cells of midgut and concentrated in the nucleus of midgut cells. In the L5D3 larvas hemocytes, BmGATA6 was detected only in cytoplasm of granular cells with low expression, but not in plasma cells. These results suggested that BmGATA6 gene plays an important role in the growth and development of silkworm. 3. Study on the mechanism of apoptosis induced by BmGATA6 gene in silkwormConstructed BmGATA6 over-expression vector and over-expression of BmGATA6 gene in Bombyx mori embryonic cell line BmE by cell transfection. Western Blotting results showed that BmGATA6 protein was expressed after transfection for 24 h, and its protein content increased with time. Fluorescence microscopy showed that cells began to appear the phenomenon of fragmentation after transfection for 48 h, after transfection for 72 h and 96 h, the cell debris increased significantly. Cell apoptosis was detected by DAPI and Tunel staining, respectively. The results of DAPI staining showed that the nuclear structure of the cells which are over-expressed BmGATA6 are loose, and the chromatin distribution show irregular shape. Tunel staining results showed that the rate of Tunel staining is significantly higher than the control group. The expression of apoptosis related genes in the silkworm was detected by fluorescence quantitative PCR. The results showed that the expressions of those genes are significantly up-regulated. Apoptosis protein Caspase3/7 activity assay showed that the activity could be significantly increased by over-expressing of BmGATA6. These results showed that BmGATA6 could induce the apoptosis of silkworm cells.BmGATA6 antibody was used for immunoprecipitation, and the obtained products were identified by mass spectrometry analysis. The results showed that there were eleven proteins which may interact with BmGATA6. According to the molecular weight and function and other factors, we can finally determine the PARP, which can promote the apoptosis of the cell. Immunofluorescence co-localization showed that PARP and BmGATA6 protein were expressed in cell nucleus. Further immunoprecipitation verified that BmGATA6 could interact with PARP protein. These results suggested that BmGATA6 induce apoptosis may be triggered by the interaction with the PARP protein. 4. FOG protein inhibits BmGATA6 to induced apoptosisCompared with silkwormovarian cell line BmNs, in which the FOG factor was highly expressed, apoptosis was more rapid and obvious in BmE cell line, in which BmUsh was not expressed. BmGATA6 and BmUsh were both over-expressed in BmE cell line, and the result showed that apoptosis induce by BmGATA6 is significantly inhibited. The result of Tunel staining showed that the numbers of Tunel opsitive cells which BmGATA6 and BmUsh genes are over-expressed simultaneously are significantly decreased compared with that in only expressed BmGATA6 gene, and the activity of Caspase3/7 is significantly decreased, too. The results showed that the FOG factor BmUsh of silkworm could inhibit the apoptosis induced by over-expression of BmGATA6.By prokaryotic expression and protein purification, we obtained a recombinant BmGATA6 protein. Far-western proved that BmGATA6 could interact with BmUsh protein, and its interaction region is at the N-terminal of BmGATA6. Further co-immunoprecipitation experiments also confirmed that the FOG factor BmUsh can combined with PARP under the condition of co-expression of BmGATA6, but it can not combined with PARP in the absence of overexpression of BmGATA6. The results indicated that BmUsh interactd with PARP indirectly through BmGATA6 binding. Fluorescence quantitative PCR detection of the expression of BmUsh and BmGATA6 in silkworm during different stages midgut and hemocytes revealed that BmGATA6 was expressed at a high level in the midgut tissues, while the expression of BmUsh expressed was higher in hemocytes. However, the two genes had similar expression pattern that they were both highly expressed in molting stages and metamorphosis development stages. The results suggested that the silkworm FOG factor BmUsh may regulate growth and metamorphosis of silkworm cooperate with BmGATA6 by interacting with BmGATA6. 5. Function of BmGATA6 gene in silkwormCombining the conclusion and analysis, we hypothesized that the BmGATA6 in the silkworm was up-regulated by some growth factors or hormones(such as 20E) during the growth and metamorphosis development, and BmGATA6 induced apoptosis by interacting with PARP or other regulatory mechanisms that were still unknown during the process of programmed cell death. When the degree of apoptosis reached the expected level, the FOG factor BmUsh would directly interact with BmGATA6 to inhibit the apoptosis induced by BmGATA6, and maintained the regular growth and metamorphosis development process of the silkworm.