Self Activation And Interaction Protein Analysis Of MdHB1 Transcription Factor In Apple

MdHB1 is the transcription factors of MdACO1,which belongs to(Homeobox protein)HD-Zip I transcription factors. HD-Zip I has closely relateship with plant abiotic stress, leaf morphogenesis, abscisic acid response, mature aging regulation and other physiological activities.This study was aimed to screen proteins interacting with MdHB1 and provide a clue to reveal the molecular network. It introduced a chance for us to elaborate the molecular mechanism of protein networks during the process of apple growth and development. And laid a sound foundation for studying the function of MdHB1 in apple fruit.1. Total RNA was extracted from peel of Gala apples and purified with removal of DNA using SDS- high purity phenol method. SMART technology were applied to synthesis double stranded cDNA with the extracted RNA, of which the short segment cDNA was removed by CHROMA SPIN TE-400 column. The rest high-quality cDNA and linearized vector pGADT7-Rec building were therefore co-transformed in yeast Y187 yeast to build the two-hybrid library. the quality of the library was tested,The library titer was 2.9×107 pfu ? mL-1, and the recombination rate was 99%. The cDNA fragments inserted were distributed from 300 bp to 2000 bp, mostly around 500 bp.2.Two fragments of MdHB1 whose length are 1000 bp, 750 bp were cloned, and then connected to pMD-19 T simple after this process, PCR was proceeded with a pair of primers containing enzyme sites(EcoR I/BamH I) to gain target sequence, and then combined with pGBKT7, By the identification of double enzyme digesting and sequence analysis, the bait expression vector pGBKT7-MdHB1, pGBKT7-MdHB1-delta CS1 were confirmed.The bait vector MdHB1 was constructed and introduced into yeast Y2 HGold by PEG/LiAC mediated transformation method. The self-activation and cytotoxicity of MdHB1 were detected in this paper. The transcription activity domain of the MdHB1 was analyzed through detecting the transcription activity of the N-terminus excluding CS1 domain of the MdHB1. Transformed yeast( pGBKT7-MdHB1) plaque growth rate and transformed into yeast(pGBKT7)plaque growth rate is quite, showing that the bait vector has no cytotoxicity The pGBKT7-MdHB1 yeast could grow on SD/-Trp and SD/-Trp/x-α-gal/AbA plates suggesting that MdHB1 protein had obvious self-activation activity. Vectors of pGBKT7-MdHB1-ΔCS1 were successfully constructed and transformed into yeast Y2 HGold, respectively. The pGBKT7-MdHB1-ΔCS1yeast was colorless on SD/-Trp/x-α-gal/AbA plates, So the N-terminus of MdHB1( excluding with transcription activity of CS1 structure domain) has no self-activation activity and toxicity.3. Our apple cDNA library by the yeast two-hybrid system were screened for pGBKT7-MdHB1-ΔCS1 without self-activation. The positive clones were screened on SD/-Ade/-His/-Leu/-Trp / x-α-gal/AbA plates, 24 blue dots identified by PCR and sequenced, and function of the interaction proteins were analyzed using NCBI database. Finally, we gained 9 protein. Function annotation showed that these candidate interacting proteins were related to photosynthesis, defense reaction and resistance.