Homotypic and Heterotypic Interactions of SatBaMV-encoded P20 Protein / 生物化学与生物物理进展

Satellite RNA ofBaMV (satBaMV), a single-stranded positive-sense RNA of 836 nucleotide [excluding 3′ poly(A) tail]containing a single open reading frame which encodes a nonstructural protein of 20 ku (P20), depends on BaMV for its replication and encapsidation. P20 is a nucleic-acid-binding protein, which facilitates the long distance movement of satBaMV in plants.Protein-protein interactions were analyzed by a bacterial two-hybrid system and pull-down assays to investigate whether P20 could self-associate and/or interact with the helper virus proteins. Self-interaction of P20 was the strongest among the viral protein-protein interactions detected in vivo and in vitro. Significant interactions of P20 with methyltransferase (MET) and capsid protein (CP) of BaMV were evidenced. Interactions among triple gene block protein 1, 2 and 3 (TGBp1, TGBp2 and TGBp3) of BaMV were also significant. BaMV CP also exhibited a strong self-interaction and strong affinity to TGBp1, TGBp2 and TGBp3. By deletion analysis,the minimal region delineated for the self-interaction of P20 was the N-terminal 15 amino acids which include the RNA binding motif of P20. N-terminal deletion resulted in the loss of self-interaction of P20. Deletion analysis also confirmed the importance of β-sheet structure of P20 in the P20-P20 interaction. P20 showed significant interactions with two host (Nicotiana benthamiana) proteins,cytochrome-C reductase and β-tubulins. The homotypic and heterotypic interactions of BaMV proteins and P20 in vivo thus suggest that the protein-protein interactions may directly exert an effect on the BaMV and satBaMV RNA movement as a protein complex in the infected host plants.

Tissue culture system of Cynanchum otophylum in genetic transformation / 中草药

Objective To establish the tissue culture system of Cynanchum otophylum in genetic transformation. Methods Asepsis seedling was set up by using different explant parts, disinfectors, and disinfecting time. The callus was induced, breeded, and differentiated by using different media, different hormone categories and combinations, and different hormone concentrations of 2,4-D and KT. Results It was the best method that asepsis seeding was built up by using seed as the explant and using 10% NaClO to treat the seed for 20—30 min or using 0.2% HgCl2 to treat the stem for 3 min. The seed, root, stem, and leaf of C. otophylum can form callus easily. The 2,4-D was important in forming callus. C. otophylum was more sensitivity to KT. The effect was better when KT was used to induce callus or adventitious buds. MS+2,4-D 1.0 mg/L+KT 1.0 mg/L was the better medium to induce callus to form. The callus cant be induced to form adventitous buds without reference to using seed or root, stem, and leaf as the explant, without reference to using hormone 6-BA or KT, ZT, 2ip. Gentamicin 100 ?g/mL is the optimum pressure in genetic transformation of C. otophylum. Conclusion The seed or stem is the ideal material bombarded by particle gun that is induced to form callus for 14 d in the media MS+2, 4-D 1.0 mg/L+KT 1.0 mg/L and without forming adventitious buds.