Expression And Functional Characterization Of GhADF7 Gene In Cotton (Gossypium Hirsutum)

Cotton is one of the mainstays of economy in China.It is of significance to clone genes related to reproduction and to elucidate the molecular basis underlining reproduction.Actin is an important protein in eukaryotic cells and constitutes actin filament,which regulates several vital cellular processes,such as mitosis,meiosis, chromosome movement,organelle movement,cytoplasmic streaming.Because actin deploymeriting factors(ADFs) regulate actin dynamics in a variety of cellular processes, ADFs are thought to affect many aspects of physiological activities of cells.However,up to now,most studies about ADF genes were focused on animals,there is few report on ADF genes that function in cotton anther development..Previously 8 ADF genes were isolated from cotton cDNA libraries in our lab.One gene expressed specifically in cotton anther was chosen for further research,which we designagted GhADF7.The main results are as follows:1.GhADF7 protein structure had high conservationSequence analysis revealed that GhADF7 encoded a protein with 139 amino acids containing a PIP2/actin binding site.A BLAST search revealed that GhADF7 deduced amino acid sequence had significant sequence similarity to other pollen-specific ADF proteins.These amino acid sequences share a 66 to 80%identity.The 8 ADF proteins in cotton have a 56 to 79%sequence similarity,for example,GhADF7 shares 79%identity with GhADF2 at amino acid level and 69%at nucleotide level.The evolutional conservatism and amino acid sequence homology of ADF gene were confirmed by the analyses of protein structure and phylogenetic evolution.2.GhADF7 expressed specifically in cotton antherReal-time RT-PCR technique was employed to analyze the expression pattern of GhADF7 gene.The results showed that the GhADF7 gene was expressed specifically in anther while no or little transcription was detected in other organs analyzed in this study. With the development of anther,the transcripts became more aboundant indicating GhADF7 is anther-preferential and also developmentally regulated.To further investigate the tissue-preferential and developmental regulated expression of GhADF7,a 1 kb fragment upstream of the GhADF7 translation start codon(ATG) was isolated by Genome walking,the GUS reporter cassette was constructed designated as GhADF7∷GUS.The GhADF7∷GUS construct was introduced into tobacco by Agrobacterium tumefaciens-mediated transformation.Histochemical assays demonstrated GUS expression driven by the promoter was specific in anthers.Furthermore,after paraffin-embedded sectioning,high level of GUS expression was detected in the pollen, especially at the tip oftransgenic pollens that germinated in vitro.3.Yeast cells overexpressing GhADF7 gene showed abnomal F-actin filament Overexpression vector was constructed by cloning the GhADF7 gene into the plasmid pREP5N and was transformed into Schizosaccharomyces pombe.After induction, the transformed yeast cells are longer than control cells;Further DAPI stanining results showed that about 78%of the transgenic yeast cells were binuclear,some even have four nucleis.In addition,the results of TRITC- phalloidin staining displayed that over-expression of the GhADF7 gene could affect the formation of actin ring and disturb cytoplast division,while have no effect on nucleus division.To further investigate the function of GhADF7 in cotton,a RNAi vector was constructed and introduced into cotton.After ten month of transformation,several lines of RNAi plants were obtained.Phenotypical analyses of these transgenic plants are under way.