Molecular cloning and biochemical characterization of alpha- and beta-tubulin from potato plants (Solanum tuberosum L.).
Few studies have investigated microtubules from plants that host pathogenic fungi. Considerable efforts are underway to find an antimitotic agent against plant pathogens like Phytophthora infestans. However, screening the effects of antifungal agents on plant tubulin in vivo or using purified native microtubule in vitro is a time consuming process. A recombinant, correctly folded, microtubule-like structure forming tubulin could accelerate research in this area. In this study, we cloned full length cDNAs isolated from potato leaves using reverse-transcribed polymerase chain reaction (RT-PCR). Solanum tuberosum (Stub) alpha-tubulin and beta-tubulin were predicted to encode 449 and 451 amino acid long proteins with molecular masses of 57 kDa and 60 kDa, respectively. Average yields of alpha- and beta-tubulin were 2.0-3.5 mg l(-1) and 1.3-3.0 mg l(-1) of culture, respectively. The amino acids, His6, Glu198, and Phe170 involved in benomyl sensitivity were conserved in Stub tubulin. The dimerization of tubulin monomers was confirmed by western blot analysis. When combined under appropriate conditions, these recombinant alpha- and beta-tubulins were capable of polymerizing into microtubules. Accessibility of cysteine residues of tubulin revealed that important ligand binding sites were folded correctly. This recombinant tubulin could serve as a control of phytotoxicity of selected antimitotic fungicide compounds during in vitro screening experiments.
Koo
Bon-Sung
BS
Fermentation and Food Processing Division, Department of Korean Food Research for Globalization, National Academy of Agricultural Science, Suwon 441 857, South Korea.
Kalme
Satish
S
Yeo
Soo-Hwan
SH
Lee
Su-Jae
SJ
Yoon
Moon-Young
MY
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D019295:Computational Biology; D003545:Cysteine / Q000235:genetics; D019281:Dimerization; D017343:Genes, Plant; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012984:Software; D011198:Solanum tuberosum / Q000235:genetics*; D014404:Tubulin / Q000235:genetics*
D014404:Tubulin; D003545:Cysteine