Cloning And Expression Characterization Of GhADF In Cotton

Actin cytoskeleton is critical for the development and morphogenesis in plant cells such as pollen tubes,root hairs and fiber cells.A lot of studies showed that ADF/cofilin is essential to the function and organization of actin cytoskeleton which is necessary to cell elongation and tip growth. To investigate gene structure, conservation and function in cotton, we isolated seven ADF/cofilin genes which belong to a gene family from cotton cDNA library and do some analysis.1. Sequence analysisAll the 11 ADF/cofilin genes from cotton (Gossypium hirsutum ) and other plants contain about 420 bp open-reading-frame which encode a protein with 140 amino acids or so and share high homology each other. In this study, the 7 cotton ADFs deduced from the isolated GhADF genes have diverged into 4 subclasses, GhADF218 belong to a single clade.There are higher similarity among GhADF218 and those pollen-specific ADF genes from other plants in the DNA and protein sequences, suggesting that those genes may have very close evolutionary relationship,which manifests that GhADF218 probably involves in the pollen tube development.2. RNA gel blot analysisRNA was extracted from young cotton tissues, we analyzed the expression patterns of GhADF218 and GhADF 105 gene by RNA gel blot, using 3'-untranslated cDNA fragments as probes. In these experiments, GhADF218 transcripts were found in anther specificly, while GhADF105 transcripts were detected almost in all organs although their expression levels in ovules, fibers, anthers and hypocotyls were relatively higher.3. DNA cloning and transformationDifferent ADF fragments were cloned into pPZP221 and pBI121 to obtain overexpression vector and supression vector respectively. Cotyledon and hypocotyl explants from Coker 312 were transformed using Agrobacterium-mediated transformation.