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Nanasato, Yoshihiko,Konagaya, Ken-Ichi,Okuzaki, Ayako,Tsuda, Mai,Tabei, Yutaka 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.3
An improved method for genetic transformation of cucumber (Cucumis sativus L. cv. Shinhokusei No. 1) was developed. Vacuum infiltration of cotyledonary explants with Agrobacterium suspension enhanced the efficiency of Agrobacterium infection in the proximal regions of explants. Co-cultivation on filter paper wicks suppressed necrosis of explants, leading to increased regeneration efficiency. Putative transgenic plants were screened by kanamycin resistance and green fluorescent protein (GFP) fluorescence, and integration of the transgene into the cucumber genome was confirmed by genomic polymerase chain reaction (PCR) and Southern blotting. These transgenic plants grew normally and $T_1$ seeds were obtained from 7 lines. Finally, stable integration and transmission of the transgene in $T_1$ generations were confirmed by GFP fluorescence and genomic PCR. The average transgenic efficiency for producing cucumbers with our method was $11.9{\pm}3.5%$, which is among the highest values reported until date using kanamycin as a selective agent.
Yoshihiko Nanasato,Ken-ichi Konagaya,Ayako Okuzaki,Mai Tsuda,Yutaka Tabei 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.3
An improved method for genetic transformationof cucumber (Cucumis sativus L. cv. Shinhokusei No. 1) wasdeveloped. Vacuum infiltration of cotyledonary explantswith Agrobacterium suspension enhanced the efficiency ofAgrobacterium infection in the proximal regions of explants. Co-cultivation on filter paper wicks suppressed necrosis ofexplants, leading to increased regeneration efficiency. Putative transgenic plants were screened by kanamycinresistance and green fluorescent protein (GFP) fluorescence,and integration of the transgene into the cucumber genomewas confirmed by genomic polymerase chain reaction(PCR) and Southern blotting. These transgenic plants grewnormally and T1 seeds were obtained from 7 lines. Finally,stable integration and transmission of the transgene in T1generations were confirmed by GFP fluorescence andgenomic PCR. The average transgenic efficiency for producingcucumbers with our method was 11.9 ± 3.5 %,which is among the highest values reported until date usingkanamycin as a selective agent.
Interaction of Tomato Mosaic Virus Movement Protein with Tobacco RIO Kinase
Hiroshi Nyunoya,Kuniaki Yoshioka,Yasuhiko Matsushita,Masahiro Kasahara,Ken-ichi Konagaya 한국분자세포생물학회 2004 Molecules and cells Vol.17 No.2
Tomato mosaic virus (ToMV) has a regulatory gene encoding a movement protein (MP) that is involved in the cell-to-cell movement of viral RNA through plas-modesmata. To identify the host cell factors interacting with ToMV MP, we used a recombinant MP probe to isolate cDNA clones from a phage expression library of Nicotiana tabacum by a far-Western screening method. One of the cDNA clones encoded an MP-interacting protein, MIP-T7, homologous to the yeast novel pro-tein kinase, Rio1p. We isolated a full-length cDNA by RT-PCR. The putative gene product was designated NtRIO, and shared 33 and 73% amino acid identity with yeast and Arabidopsis RIO kinases, respectively. In vitro analyses using recombinant proteins showed that NtRIO also interacted with a different MP de-rived from Cucumber mosaic virus. NtRIO had auto-phosphorylation activity and phosphorylated ToMV MP. Addition of recombinant tobacco casein kinase 2 resulted in a marked increase in the phosphorylation of NtRIO. The interaction between NtRIO and ToMV MP was inhibited by phosphorylation of NtRIO.