Preparation of CsHB1 polyclonal antibody and its protein dynamic changes during somatic embryogenesis in Citrus

Abstract：【Objective】This study was aimed at obtaining the high efficient protein expression of CitrusHD-ZIP II transcription (CsHB1) in Escherichia coli to prepare the polyclonal antibody, and investigatethe specificity of antibody during the callus induction of somatic embryos. After analyzing the motif anddomain of CsHB1 amino acid sequence, the prokaryotic expression vector was constructed and the ex-pressed product was obtained in E. coli. Then the polyclonal antibody was prepared by immunization. Thepolyclonal antibody was used to detect the specificity of the antibody in the prokaryotic expression systemand to detect the dynamic expression of the protein during the induction of somatic embryo by using west-ern blot analysis.【Methods】The CsHB1 sequence was predicted by sequencing and domain analyses. TheHD-ZIP-N domain and homeo domain was selected for constructing the prokaryotic expression system.The specific sequence was amplified by PCR and inserted into the p GEX-4T-1 vector. The recombinantplasmid was named as p GEX-4T-CsHB1-N. The recombinant plasmid p GEX-4T-CsHB1-N was incu-bated with 1 mmol·L^-1 PTG at 37 ℃ for 0, 2, 4, 6, 8, 10 and 12 h in LB liquid. The SDS-PAGE analysiswas then performed to detect the protein expression of CsHB1-N in the prokaryotic system. Then the pro-karyotic expression product of GST-CsHB1-N was induced with 300 mL LB liquid, and the supernatantwas collected by centrifugation at 9 000 r·min^-1 t 4 ℃ and purified with a GST-tag. After purification, the rabbits were immunized with GST-CsHB1-N to obtain the anti-CsHB1-N polyclonal antibody. Theexpression products of p GEX-4T-CsHB1-N recombinant strain and p GEX-4T-1 strain induced byIPTG were analyzed by SDS-PAGE, and the protein was transferred to the PVDF membrane to detect theanti-CsHB1-N antibody specificity in the prokaryotic expression system. The callus materials of Valenciawere cultured in a glycerol medium under light conditions for 0, 14, 28 and 42 d. The total protein was ex-tracted, and the protein concentration was determined by a Bradford assay. The specificity of the CsHB1 polyclonal antibody and its protein dynamic changes was detected in the somatic embryo induction stageof‘Valencia Orange'callus by a western blot analysis.【Results】The CsHB1 gene contained 287 aa. Theprotein was composed of HD-ZIP-N (1-101 aa) , homeodomain (128-186 aa) and HALZO (186-229 aa) .The full-length sequence CsHB1 failed to express the target protein in the prokaryotic expression system, so the fragment of 1-187 aa was selected to construct a prokaryotic expression vector. The fusion vectorp GEX4T-CsHB1-N was confirmed by PCR and restriction enzyme digestion. The recombinant plasmidwas transformed into E. coli to induce the expression of protein. About 49 ku GST-CsHB1-N protein washighly efficiently expressed and compared with the blank vector, then a large amount of induced proteinwas purified. The GST-CsHB1-N purified protein was immunized in rabbits to obtain the anti-CsHB1-Npolyclonal antibody. The anti-GST and anti-CsHB1-N were used to detect the recombinant protein in thep GEX-4T-CsHB1-N recombinant strains induced by IPTG for 2 h and 4 h, respectively. The resultsshowed that the molecular weight of the GST-CsHB1-N fusion protein was about 49 ku, which contained27 ku GST-tag protein and 22 ku CsHB1-N protein. The western blot analysis showed that the anti-CsHB1-N polyclonal antibodies could specifically identify the corresponding antigen peptides GST-CsHB1-N in the prokaryotic expression system. In the citrus callus, the CsHB1 target protein which wasabout 32 ku, consistent with the expected size, was also detected. Using the callus proteins of‘ValenciaOrange'cultured in glycerol medium under light conditions for 0, 14, 28 and 42 d, the western blot re-sults showed that the CsHB1 protein exhibited higher expression in the embryonic callus, and the expres-sion level of CsHB1 protein was decreased at first and then increased after somatic embryo induction.【Conclusion】The anti-CsHB1-N polyclonal antibody was successfully obtained. The expression of sHB1 protein in the prokaryotic expression system and somatic embryogenesis of the embryogenic callusof the‘Valencia Orange'was detected by using anti-CsHB1-N polyclonal antibodies. The antibody canbe used for the detection of target protein CsHB1 in citrus, and it was found that the CsHB1 protein exhib-ited dynamic changes during the somatic embryos induced stage.