Construction of Yeast Two-Hybrid cDNA Library and Screening of Interaction Proteins of ZmCEN in Maize
To understand how Zea mays L.centrin(ZmCEN)function,we employed the technique of yeast two hybrid system (Y2HS) to investigate the interaction between interacting-proteins and ZmCEN.In this study,using maize inbred line Zheng 58 seedlings as materials,we isolated the total RNA and synthesized the double-strand cDNAs by SMART technology.According to the CDS sequence of the ZmCEN gene,we designed the primers to construct recombinant bait vector (pGBKT7-ZmCEN) and transform yeast strain Y2HGold for screening the toxicity and self-activation ability of bait vectors.We screened the prey proteins interaction with ZmCEN.Interacting proteins NAC67 and TON1b were verified the interaction by β-galactosidase assay in vitro.The BiFC semi-molecule vectors of ZmCEN-pSPYNE and TON1b-pSPYCE were constructed to further confirm the interaction in Arabidopsis cells.Gene and Ontology (GO) enrichment analysis were performed on the screened proteins using Uniprot and KEGG online sites.The results showed that the unamplified cDNA library was consisted of 2.56 × 107 CFU independent clones,and the titer of library was 5.36 × 108 CFU/mL which met the requirements of library construction.The cDNA library was screened by bait vector after testing no toxicity and no self-activation,and 28 proteins were interacted with ZmCEN by sequencing and Blast alignment analysis.GO enrichment analysis of these proteins showed that there were 21 terms in the biological process.The ZmCEN and TON1b proteins were further verified interaction in Arabidopsis cell by BiFC technology.A high-quality cDNA library was constructed and 28 proteins were interacted with ZmCEN,which could be used to further investigate the interaction mechanisms between them.
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