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Cloning and Characterization of GhADF Genes in Cotton

Author: ZhangChengWei
Tutor: LiXueBao
School: Central China Normal University
Course: Biochemistry and Molecular Biology
Keywords: Cotton Actin depolymerization factor Vector construction Cotton transformation Gene Expression Sequence analysis
CLC: S562
Type: Master's thesis
Year: 2006
Downloads: 95
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Abstract


ADF / cofilins (actin-depolymerizing factor / cofilin) ??is the actin binding a protein family, is widely found in all eukaryotic cells. Many studies have shown that the ADF (actin-depolymerizing factor) protein actin cytoskeletal dynamics assembled an important regulator of the nuclear top of the actin cytoskeleton cell elongation growth is necessary. Therefore separation, identification cotton ADF genes, and then study its function, and to figure out their expression and regulation has important theoretical significance and application value. 1 vector and the transformation of cotton using recombinant DNA technology, ADF cloning to pPZP221 and pBI121 were constructed excessive expression vector (overexpression vector) and RNAi (supression vector), vector. PCR amplification reaction and enzyme digestion confirmed that the gene has been successfully inserted into the vector. Has been successfully These vectors transferred into Agrobacterium (Agrobacterium LBA4404), and transformed cotton. Has been a number of cotton seedlings. 2 gene expression analysis by a modified hot phenol extraction of cotton early development organizations RNA, Northern blot analysis of the expression of individual genes. Northern blot analysis showed the expressed GhADF2 the petals anthers and 10DPA of fiber in the signal strength, while weak expression signal the root, hypocotyls, 10DPA, ovule, 15DPA the ovule well 20DPA ovules. Anthers, GhADF7 only express no signal in the other tissues, indicating GhADF7 anther-specific expression. Also carried out GhADF8 the Northern hybridization, without any expression signals, may be a GhADF8 the expression level is very low, the Northern hybridization detection of less than only. 3 Sequence analysis of the separated cotton actin protein depolymerizing factor (actin-depolymerizing Factor, ADF) cDNA sequence specific primers, upland cotton (Gossypium hirsutum) leaf DNA as a template, by polymerase chain reaction (PCR) amplified the separation obtained cotton GhADF7 gene. Using DNAStar this show: GhADF7 gene coding region contains three exons and two introns, and intron position is highly conserved gene sequence analysis. The use of technologies such as bioinformatics structural GhADF2 GhADF7 and GhADF8 three genes encoding the amino acid sequence from Arabidopsis thaliana the AtADF3 and AtADF4 the amino acid sequence homology

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