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Expression and Functional Characterization of GhADF7 Gene in Cotton (Gossypium Hirsutum)

Author: XuDan
Tutor: LiXueBao；XuWenLiang
School: Central China Normal University
Course: Biochemistry and Molecular Biology
Keywords: Cotton Actin ADF Anther-specific Promoter Pollen development Overexpression of RNAi
CLC: S562
Type: Master's thesis
Year: 2009
Downloads: 79
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Abstract


Cotton (Gossypium hirsutum) is an important economic crop. Isolation and identification of cotton reproductive and developmental important genes resolve the molecular mechanism of cotton reproductive development, to improve the yield and quality of cotton has important theoretical and practical value for the cross-breeding of cotton. Actin (actin) is an important ubiquitous protein in eukaryotic cells. Of actin the cytoskeleton microfilament system, involved in mitosis, meiosis, chromosome movement, movement of organelles, the cytoplasm flow of life activities. Because the actin cytoskeleton involved in a series of physiological activity of the cells, directly regulated by actin and actin protein depolymerization factor (ADF), and thus inevitably have an important impact on these physiological activities. Most of the current research has focused on animal cells, and reports the study of plant material is relatively small. Cotton ADF genes studies have focused on the impact of cotton fiber development, the ADF in the function of the reproductive development of cotton, has not been reported. The laboratory seven ADF genes isolated and cloned from a cDNA library of cotton. Through the analysis of patterns of gene expression, we screened an ADF genes specifically expressed in anthers, named GhADF7. Study was to investigate the expression and function of genes, achieved the following results: 1. Structure of cotton GhADF7 protein is highly conserved sequence analysis showed cotton GhADF7 gene encodes a 139 amino acids of the ADF protein contains a PIP2/actin to binding sites. Similarity of 56-79% of the protein with other seven cotton ADF protein. Which, GhADF7 GhADF2 highest similarity of 79%. ADF protein and other plant anther-specific similarity GhADF7 is 66% -80%, such as similarity with NtADF2 same amino acids up to 80%, even higher than the cotton ADF within the family of the ADF protein similarity. Phylogenetic analysis confirmed that the cotton ADF protein evolutionary conservation. The 2 cotton GhADF7 gene in anther-specific expression by RT-PCR technology to further analysis of gene expression patterns GhADF7. The results show the GhADF7 cotton anther-specific expression, and the expression levels with Anthers of rising. Adjustable features for further study of a GhADF7 the tissue-specific and developmental stages, using the Genome walking method to obtain the promoter of the gene was isolated, and was constructed GhADF7 :: GUS fusion vector. Application of Agrobacterium-mediated transformation technology transformed tobacco and Arabidopsis, get a group of transgenic tobacco and Arabidopsis plants. Histochemical analysis showed that the promoter driving in GhADF7 specific expression of the GUS gene in the anther, further paraffin section analysis showed GUS signal is mainly concentrated in the pollen. In vitro germination of transgenic tobacco pollen found strong expression GhADF7 genes in pollen tube, especially in the pollen tube tip. 3 lead to excessive express cotton GhADF7 gene the yeast intracellular actin filaments (F-actin) exception will the GhADF7 overexpressed genes in yeast cells genetically modified yeast cell elongation. The DAPI staining find 78% of yeast cells dual-core or quad-core. TRITC-phalloidin staining showed that overexpression of actin ring formation GhADF7 affect yeast cells cytoplasm segmentation blocked, normal yeast mitotic emerged multicore body. In addition, we have constructed the gene RNAi vector, and transformed cotton, get the number of transgenic RNAi plants, gene function analysis has yet to be carried out.

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CLC: > Agricultural Sciences > Crop > Economic crops > Fiber crops > Cotton
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