Abstract: 【Objective】The objective of this study is to clone and overexpress BmGATA6 in Escherichia coli to obtain the recombinant protein, which can be used to immunize mice for the preparation of polyclonal anti-BmGATA6 antibody. And to provide a theoretical basis for further studying the function of BmGATA6 in silkworm (Bombyx mori). 【Method】B. mori was killed to obtain the different tissues on day 3 of 5th instar larvae and the midguts from the molting period of the 4th instar larvae to the wandering stage, and these tissues were grinded into powder in the liquid nitrogen. Tissue RNA was extracted by Trizol, and cDNA was reversely transcribed in vitro. The full-length specific primer sequences were designed according to BmGATA6 coding sequence. Using BmActin3 as a reference gene, RT-PCR was conducted to detect the expression of BmGATA6 both in the different tissues on day 3 of 5th instar larvae and the midguts from the molting period of the 4th instar larvae to the wandering stage. The prokaryotic expression primer sequences were designed, and the amplified fragment was ligated to the pET32a, which was then transformed into BL21 (DE3) E. coil to obtained fusion protein. The recombinant BmGATA6 protein was induced with 0, 0.4, 0.6, 0.8 and 1.0 mmol·L^-1 IPTG for 20 h at 16℃ or 5 h at 37℃. The optimal induction conditions were chosen to gain BmGATA6 protein, and Ni-NTA affinity chromatography was utilized to purify the BmGATA6 protein. Then the anti-BmGATA6 polyclonal antibody was obtained after immunizing mice with recombinant protein. The expression of BmGATA6 was detected by Western blot with the antibody, which was identified by ELISA. Taking paraffin sections of the midgut on day 6 of 5th instar larvae as samples, the subcellular localization of BmGATA6 protein in the midgut of B. mori was conducted by immunofluorescence.【Result】The 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. RT-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. 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.【Conclusion】Anti-BmGATA6 polyclonal antibody was prepared successfully, and preliminary analysis of its subcellular localization in the midgut tissue was done, which revealed that BmGATA6 played an important role in the regulation of physiological changes in the midgut of B. mori, and laid the foundation for further exploration of the BmGATA6 function in B. mori.

Key words: Bombyx mori, BmGATA6, prokaryotic expression, polyclonal antibody, subcelluar localization