Cloning And Characterization Of GhADF Genes In Cotton

The ADF/cofilins are a family of actin-binding proteins expressed in all eukaryotic cells so far examined.A lot of studies showed that ADF proteins are thought to be key modulators of the dynamic organization of the actin cytoskeleton which is necessary to cell elongation and tip growth. It has important theoretics significance and application value to separate and characterise the function of ADF genes from cotton and clear up their expression and regulation.1. DNA cloning and cotton 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. The results of PCR reaction and cutting with enzymes showed that the target gene was already successfully fused with vector .At present these reform vectors were transformated into Agrobacterium(LBA4404). Now I have already got the small seedling of a batch of cottons now.2. The analysis of gene expressionCotton total RNA was isolated from different tissures of cotton , different developmental period of cotton fiber and ouvle by hot-phenolic mothed. Northern hybridization experimental results showed that the expression levels of GhADF105 in petal.anther and 10DPA fiber is higher than it in root,hypocotyls,10DPA ovule, 15DPA ovule and 20DPA ovule. While GhADF218 only expresses in anther. At the same time we also make use of northern blot to analysis the expression of GhADF220, but its transcripts can not be detected in all organs. The reason may be the low expression of GhADF220.3. Sequence analysisThe special primers were designed by the ADF218 cDNA isolated fromcotton.Using the DNA of Gossypium hirsutum leave as template, GhADF218 gene were obtained by PCR. The gene's sequence was analyzed by means of DNAStar. The coding region of the GhADF7218gtne contain three extrons and two introns, and the position of the intron is highly conserved. The structure homology and molecular evolution of GhADF2, GhADF7 and ADF8 from cotton and AtADF3, AtADF4 from Arabidopsis thaliana were analyzed by means of bioinformatics. Each of the genes deduced an ADF protein containing 139 amino acid residues. The three ADF genes share 73 - 87% identity at amino acid level. Analysis of evolution relationship revealed that GhADF2 is closed to PeADF2, sharing 89% homology each other. GhADF7 is more closely related to LIADF and ZmADF than to NtADFl and NtADF2. Their homology is between 75 % -79 %. GhADF8 have very close relative relatitionship. The evolutional conservatism and amino acid sequence homology of ADF gene were confirmed by the analyses of protein structure and phylogenetic evolution.