Transformation Of Tobacco With Arabidopsis Thaliana MAP65-6 And TUA-6

Cell morphogenesis is the essential condition in plant growth and development, and cytoskeleton plays a key function in cell morphagenesis. Cytoskeleton contains microtubules, microfilament and intermediate filament, of which microtubules play an important role in cell division, cell differentiation, cell morphous, celluar material transportation and other physiology activity. Microtubule array in plant cell is different from animals', such as cortical microtubule, proprephase band, spindle microtubule and phragmoplast microtubule. Microtubule array transforms in order during plant cell morphogenesis and needs participation of microtubulin and microtubule-associated proteins.In this study, MAP65-6-GFP, GFP-TUA-6 and TUA-6-GFP were transferred into tobacco plants by Agrobacterium mediated method.We perfected the method of genetic transformation for tobacco-NC89. The handling steps:First, tobacco cotyleton were pre-cultured for 2 d in darkness; second, the tobacco cotyleton infected by Agrobacterium for 5 min; then, co-cultured for 2 d in darkness; and then, deprived of Agrobacterium with reasonable and effective way; finally, transferred them to the callus induced medium, differential medium, and rooting medium by turns. In total,88 transformed lines were regenerated.Of 36 MAP65-6-GFP transformed tobacco lines,17 lines were determined to be PCR positive, and transformation efficiency was 47%. Three lines in 5 lines analyzed by Southern blot had hybridization signal, which indicated that MAP65-6-GFP sequence has been inserted into the genome of tobacco lines. By laser scanning confocal microscopy, we observed that MAP65-6-GFP in root localized mainly in cytoplasm and cortical microtubules.Of 33 GFP-TUA-6 transformed tobacco lines,12 lines were determined to be positive by PCR analysis and transformation efficiency was 36%. Three lines in 5 lines analyzed by Southern blot had hybridization signal, which indicated that GFP-TUA-6 sequence was been inserted into the genome of tobacco lines. By laser scanning confocal microscopy, we observed that GFP-TUA-6 in root localized mainly in cortical microtubules.Analysis on 19 TUA-6-GFP transformed tobacco lines by PCR,4 lines were determined to be positive, and transformation efficiency was only 21%. three lines had Southern hybridization signal, which indicated that TUA-6-GFP sequence was been inserted into the genome of tobacco lines. By laser scanning confocal microscopy, we observed that TUA-6-GFP in root localized mainly in cytoplasm and cortical microtubules.These MAP65-6-GFP, GFP-TUA-6 and TUA-6-GFP transgenic lines with intensive and stable fluorescence will provide a base for studying the regulation of MAP65-6 on microtubule array. And also GFP-TUA-6 and TUA-6-GFP transgenic lines could be used to further study the dynamics mechanism of cortical microtubule in cell morphogenesis as probes in vivo.