Liu Jinyuan

Preparation of the selenomethionine derivative of tabtoxin resistance protein

In order to obtain phase information for the X-ray diffraction of tabtoxin resistance protein (TTR) crystal using the MAD phasing method, a selenomethionine (Se-Met) derivative of TTR was overexpressed inE. coli strain M15, with pQE-30 plasmid, through IPTG induction in M9 medium containing Se-Met. The product was purified to an estimated homogeneity of greater than 95% according to SDS-PAGE, by a Ni-NTA metal affinity followed by a Mono Q anion exchange column chromatography. The successful substitution of Se-Met for methionine (Met) was confirmed by MALDI-TOF and ESI-Quadrupole Mass Spectrometry analysis. The derivative crystal was obtained using similar conditions as those for the native.

Molecular Characterization of a Cytosolic Malate Dehydrogenase Gene( GhcMDH1 ) from Cotton

Malate dehydrogenase(MDH) is a key enzyme that catalyzes the reversible oxidation of oxaloacetate to malate and plays an important role in the physiological processes of plant growth and development. However, cyto-solic malate dehydrogenase(cMDH), which is crucial for malate synthesis in the cytosol, still has not been extensively characterized in plants. Here, we isolated a cytosolic malate dehydrogenase gene, designated as GhcMDH1, from Gossypium hirsutum and characterized its possible molecular function in cotton fiber. The cloned cDNA of GhcMDH1 is 1520 base pairs in length, and has an open reading frame of 999 base pairs, encoding for 332 amino acid residues with an estimated molecular weight of 35580 and pI of 6.35. Sequence alignment showed that the de-duced amino acid sequence of GhcMDH1 protein shared a high similarity to other plant cMDHs. Confocal and im-munological analysis confirmed that GhcMDH1 protein was subcellularly localized to the cytosol. Quantitative real-time polymerase chain reaction(PCR) revealed that GhcMDH1 was constitutively expressed in all vegetative cotton tissues, with slightly lower levels in roots than stems and leaves. Interestingly, the transcripts of GhcMDH1 were detected in 5―25 d post anthesis(DPA) fibers and highly abundant at 15 DPA fibers. The total MDH activities and malate contents of cotton fibers were positively correlated with the fiber elongation rates, suggesting that GhcMDH1 may function in malate synthesis in fast fiber elongation. In agreement with this suspicion, the recombi-nant His-GhcMDH1 protein mainly drives the reaction towards malate generation in vitro. In conclusion, our mole-cular characterization of the GhcMDH1 gene provides valuable insights to further investigate the malate equilibrium in cotton fiber development.

Observation of the in-vivo Reporter of Green Fluorescent Protein in a Plant Root by Scanning Near-Field Optical Microscopy

An in vivo reporter of green fluorescent protein (GFP) in a living plant root has been imaged by scanning near-field optical microscopy (SNOM) in the transmission mode. The exciting light is 488 nm wavelength of the argon ion laser and the bandpass filter (514+/-10) nm is put into the detecting optical pathway. The results indicate that, in the living plant cells, the GFPs gather together and form an area of 2-4 mum, rather than being individually distributed. The transmission coefficient of the eigenfunction is incorporated into Bethes theoretical model modified by Grober, and the near-field excited light intensity is calculated along the fibre probe axis (z-axis) in the air medium and biological medium. Based on this, along the z-axis direction of the GFP detected in the sample, numerous GFPs locate near the epidermal cells wall (in the range of 0-38 nm) in the living root. The experiments show that SNOM has an advantage of optical nanometre-scale resolution along the z-axis.