Antibody Preparation And Functional Analysis Of BmGATA6 In The Midgut Of Silkworm,Bombyx Mori

Bombyx mori as a important Lepidoptera model organisms, a large number of cell apoptosis process happening in the metamorphosis, is an ideal material for the study of apoptosis. In addition, found that increase of GATA6 will promote the expression of Bax protein in the mammals, causing cell apoptosis. The study in zebrafish found that downregulation of GATA6 will cause the functional defects of intestinal smooth muscle, but also cause the abnormal development of the intestinal tract. The Bombyx mori is one member of the holometabolous class of inse, which through several molting period. During Larva-pupae Metamorphosis, a large number of midgut cell have a autophagy and apoptosis, which is also continue to produce. This process is a precise regulation of the molecular network.GATA is a conserved nucleic acid sequence in gene promoter. Many transcriptional regulatory proteins that identify GATA sequences have been identified, which have a function of regulating transcriptional activity. Therefore, these proteins called GATA transcription factor. Three GATA transcription factors have been reported, including BmGATAα、BmGATAβ and BmGATA6. The function of BmGATAα has not been reported. Drevet reported that BmGATAβ formate three shear body by selective cutting: BmGATAβ1 expressed in silkworm gonad specifically; BmGATAβ2 expressed in the tissues of the larvae and pupae; BmGATAβ3 only expressed in the pupal stage follicles. The function of BmGATAβ1 is related to the formation of follicle cells and the expression of the chorionic genes, but the function of the other 2 spliceosomes has not been reported. Only in the NCBI contains a sequence information of BmGATA6. So we have a preliminary study of the function of BmGATA6. The main results of this study are as follows: 1. Cloning and analysis of BmGATA6The cDNA of BmGATA6 was 4 011 bp. The ORF of BmGATA6 was 1 974 bp, encoding 657 amino acids. The cDNA of BmGATA6 was comprised of 205 bp 5′UTR and the 1 832 bp 3′UTR. BmGATA6 was located on chromosome 15 in B. mori genome, and the full-length was 12 326 bp. BmGATA6 was a gene with multiple exons structure, including 8 exons and 7 introns. The predicted protein sequence contained two conserved zinc-finger domains, which were located in 470-521 and 531-582 amino acid regions. The sequence alignment and The phylogenetic tree showed that BmGATA6 was very and was closer to the GATAc of Danaus plexippus. 2. The mRNA expression pattern of BmGATA6 in silkwormRT-PCR results showed that the BmGATA6 was highly expressed in the midguts on day 3 of 5th instar larvae, while the expression was low in the Malpighian tubule, and no BmGATA6 expression was detected in other tissues. Besides, RT-PCR results showed that the BmGATA6 was continually expressed in midguts from the molting period of the 4th instar larvae to the wandering stage. 3. Prepare Anti-BmGATA6 polyclonal antibodySelect the specific fragment of N terminal 944 bp in the BmGATA6 coding frame. Construction of prokaryotic expression vector of BmGATA6, it was found that the recombinant BmGATA6 protein mainly existed in the supernatant at 16 ℃, or in inclusion bodies at 37℃. The recombinant BmGATA6 protein was best induced with 0.6 mmol?L-1 IPTG for 20 h at 16 ℃, then purified using Ni-NTA affinity chromatography. To obtain the anti-BmGATA6 polyclonal antibody, mice was immunized repeatedly with pure protein. ELISA indicated that BmGATA6 antibody titer was 1﹕128 000. Western blot demonstrated that anti-BmGATA6 polyclonal antibody, which was prepared by ourself, could detect BmGATA6 protein specifically. BmGATA6 protein molecular weight was about 75 kD, consistent with prediction. Immunofluorescence results showed that BmGATA6 was highly expressed in midgut cells of B. mori, and it was located in nucleus. 4. The function of BmGATA6 in the midgutBmGATA6 protein expression analysis found, BmGATA6 highly expressed in the midgut, other tissues express low, and the highest expression of sterile pupae in intestinal development different periods of time. We use dsRNA interference BmGATA6 in silkworm mature period. It was found that, midgut BmGATA6 expression is a clear downward after interference 24 h. BmGATA6 midgut immunofluorescence found in the intestinal intestinal expression was significantly down-regulated the expression of BmGATA6. TUNE detected that BmGATA6 down regulated expression of intestinal wall cells was not detected on TUNEL signals, while in the amorphous mass of exfoliated cells from the inside of the intestinal wall has a large number BmGATA6 expression. And two days after interference BmGATA6 found that the normal development of the larvae in the control group, the emergence of the larvae lethality in the experimental group, and then the experimental group were all died.