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

XIE Xingnan; LAI Xiaona; ZHAN Shuang; CHENG Laichao; XU Quanquan; GE Xiaoxia

doi:10.13925/j.cnki.gsxb.20170022

Abstract：[Objective] This study was aimed at obtaining the high efficient protein expression of Citrus HD-ZIP Ⅱ transcription (CsHB1) in Escherichia coli to prepare the polyclonal antibody,and investigate the specificity of antibody during the callus induction of somatic embryos.After analyzing the motif and domain of CsHB1 amino acid sequence,the prokaryotic expression vector was constructed and the expressed product was obtained in E.coli.Then the polyclonal antibody was prepared by immunization.The polyclonal antibody was used to detect the specificity of the antibody in the prokaryotic expression system and to detect the dynamic expression of the protein during the induction of somatic embryo by using western blot analysis.[Methods]The CsHB 1 sequence was predicted by sequencing and domain analyses.The HD-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 pGEX-4T-1 vector.The recombinant plasmid was named as pGEX-4T-CsHB1-N.The recombinant plasmid pGEX-4T-CsHB1-N was incubated with 1 mmol· L-1 IPTG at 37 ℃ for 0,2,4,6,8,10 and 12 h in LB liquid.The SDS-PAGE analysis was then performed to detect the protein expression of CsHB 1-N in the prokaryotic system.Then the prokaryotic expression product of GST-CsHB1-N was induced with 300 mL LB liquid,and the supernatant was collected by centrifugation at 9 000 r· min-1 at 4 ℃ and purified with a GST-tag.After purification,the rabbits were immunized with GST-CsHB 1-N to obtain the anti-CsHB 1-N polyclonal antibody.The expression products of pGEX-4T-CsHB1-N recombinant strain and pGEX-4T-1 strain induced by IPTG were analyzed by SDS-PAGE,and the protein was transferred to the PVDF membrane to detect the anti-CsHB 1-N antibody specificity in the prokaryotic expression system.The callus materials of Valencia were cultured in a glycerol medium under light conditions for 0,14,28 and 42 d.The total protein was extracted,and the protein concentration was determined by a Bradford assay.The specificity of the CsHB 1 polyclonal antibody and its protein dynamic changes was detected in the somatic embryo induction stage of ‘Valencia Orange‘ callus by a western blot analysis.[Results] The CsHB 1 gene contained 287 aa.The protein was composed of HD-ZIP-N (1-101 aa),homeodomain (128-186 aa) and HALZO (186-229 aa).The full-length sequence CsHB 1 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 vector pGEX4T-CsHB1-N was confirmed by PCR and restriction enzyme digestion.The recombinant plasmid was transformed into E.coli to induce the expression of protein.About 49 ku GST-CsHB1-N protein was highly efficiently expressed and compared with the blank vector,then a large amount of induced protein was purified.The GST-CsHB1-N purified protein was immunized in rabbits to obtain the anti-CsHB 1-N polyclonal antibody.The anti-GST and anti-CsHB 1-N were used to detect the recombinant protein in the pGEX-4T-CsHB1-N recombinant strains induced by IPTG for 2 h and 4 h,respectively.The results showed that the molecular weight of the GST-CsHB1-N fusion protein was about 49 ku,which contained 27 ku GST-tag protein and 22 ku CsHB1-N protein.The western blot analysis showed that the antiCsHB1-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 was about 32 ku,consistent with the expected size,was also detected.Using the callus proteins of ‘Valencia Orange’ cultured in glycerol medium under light conditions for 0,14,28 and 42 d,the western blot results showed that the CsHB1 protein exhibited higher expression in the embryonic callus,and the expression 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 CsHB1 protein in the prokaryotic expression system and somatic embryogenesis of the embryogenic callus of the ‘Valencia Orange’ was detected by using anti-CsHB1-N polyclonal antibodies.The antibody can be used for the detection of target protein CsHB 1 in citrus,and it was found that the CsHB 1 protein exhibited dynamic changes during the somatic embryos induced stage.