Dissertation > Excellent graduate degree dissertation topics show

Cloning and Functional Analysis of A New Cotton Gene GhDr1 Related in Resistense To Abiotic Stresses

Author: DingZhongTao
Tutor: ZhangRui
School: Chinese Academy of Agricultural Sciences
Course: Crop Genetics and Breeding
Keywords: Upland cotton GhDr1 Alternative splicing Expression profiling Subcellular localization Physiological experiments
CLC: S562
Type: Master's thesis
Year: 2010
Downloads: 120
Quote: 0
Read: Download Dissertation

Abstract

Drought, soil salinization, climate anomalies chilling injury due to heat injury abiotic stresses stress is seriously affecting the yield and quality of crops. Plant stress when the injury can be reduced through its own stress tolerance to abiotic stress caused by abiotic stresses, plant cells stimulated by abiotic stress signal, the signal transduction pathways ultimately mobilize functional gene expression completed. Protein expression changes in the signal transduction pathway may affect plant tolerability Stress. MAPK, CDPK, CIPK signal path discovery involving protein phosphorylation cascade amplification, signal transduction downstream gene expression. By means of genetic engineering regulation of signal transduction pathways on gene expression, and has broad prospects for improved stress resistance in plants like. This study on the basis of in the the laboratory cloning cotton resilience gene fragments, the of cotton resilience gene cloning and preliminary functional analysis. Achieved major results are as follows: 1. In the resilience related gene GhDr1 346bp gene fragments on the basis of genomic BAC library by screening the GhDr1 positioning single BAC clones; TAIL-PCR amplification GhDr1 the 5 'end and 3 'flanking sequence, the the 7037bp genome sequence of GhDr1 final. By RT-PCR and TAIL-PCR amplification obtain GhDr11913bp of cDNA sequence. 2. Analysis to prove GhDr1 contains four exons and three introns, and the transcription start site is located at the upstream of the ATG 492bp. The second intron GhDr1 exist phenomena of alternative splicing, four splice isoforms generated by alternative splicing. The differences in the amino acid sequence corresponding to the protein encoded by the splice isoforms peptide region contains more can be phosphorylated adjustable serine and threonine, and affecting the protein secondary structure proline, these differences lead to the predicted phosphorylation sites and secondary structure changes. GhDr1 the promoter region is located in the 5'-UTR region and the transcription start site upstream of about 1.5kb the within, the cis-acting element analysis prove GhDr1 the promoter region there are many adversities and signal transduction regulatory elements. 3. Bioinformatics analysis found: GhDr1 the coding region of a total length of 1485bp, encoding 494 amino acid residues, stable negatively charged acidic protein; GhDr1 protein secondary structure α-helix and random coil, respectively, accounting for 40.69% and 44.94%; the amino acid sequence of GhDr1 more hydrophilic amino acids and the presence of concentrated hydrophilic region, no obvious transmembrane helices; The subcellular localization forecast GhDr1 located in the cytoplasm of most likely; homology alignment The results showed that the GhDr1 and castor, grapes, Populus trichocarpa and Arabidopsis protein homology highest; the the functional domains predict found GhDr1 exist tyrosine kinase II phosphorylation sites, N-myristoylation site, protein kinase C phosphorylation sites, zinc finger class function modules, MAPK phosphorylation sites and MAPK interaction sites; GhDr1 many involved in plant stress responses and signal transduction in which chromosomal regions homologous genes in Arabidopsis and grape pathway of functional proteins. To predict GhDr1 phosphorylation cascade of the MAPK signal transduction path may be adjusted. 4 expression profiling showed that GhDr1 each growth period in the cotton has its expression in the organ. The cotton GhDr1 relative expression of the highest organ to mature leaf, the amount of expression in the radicle the radicle and mature root of the root class organization is relatively stable, the highest overall amount of GhDr1 expression in mature roots, stems, leaves and flowers. Building transient expression vector 35S :: 35S :: the p16318 the-GhDr1-hGFP transiently expressed in onion epidermal cells by biolistic transformation. The fluorescence signal of the fusion protein in onion epidermal cells more evenly distributed in the cytoplasm, it is determined that the GhDr1 cytosolic protein. 6. The building GhDr1 gene pBIGhDr1GUS plant dual expression vector, electroporation into Agrobacterium floral dip method transformed Arabidopsis to get transgenic plants and improved into Arabidopsis floral dip method, the conversion efficiency to about 40% . T1 generation of resistance screening PCR identification, GUS staining and T2, T3 generations separating identification to turn cotton GhDr1 gene than the screening of the final Arabidopsis strains homozygous transgenic plants. Real-Time PCR analysis of two ultra-high expression of homozygous lines, two high expression and 10 low expression in Arabidopsis. 7. Selected representative low expression in lines 38, 62 and high expression lines 24 and ultra-high amount of expression lines 61, the combination of WT and order and have been identified homozygous mutant analysis GhDr1 resilience physiological functions. The physiological experiment results show that: the phenotype of transgenic lines under normal culture conditions better than the wild-type and mutant; exogenous ABA plates turn GhDr1 low amount of expression lines insensitive ultra-high amount of expression lines sensitive; Salt Stress turn GhDr1 low expression level of Arabidopsis more sensitive strains, and turn the GhDr1 high expression strain insensitive; ABA plates germination rate of Arabidopsis thaliana strains in turn GhDr1 gene in wild-type and mutant strain the NaCl plates germination rate lower than the wild-type and mutant strains. This shows GhDr1 did participate resilience physiological processes, and its expression levels in different transgenic lines enables sensitivity to abiotic Stress reversed. In summary, this study first cloned genome sequences and promoter sequences of related the gene GhDr1 the cotton resilience found GhDr1 produce at least four splice isoforms; analysis a GhDr1 expression profiles and subcellular localization characteristics; through the GhDr1 genes transferred to the model plant Arabidopsis overexpressing homozygous transgenic Arabidopsis lines resilience physiological phenotypes finalize GhDr1 participate in adversity physiological processes. In this study, stress resistance can be improved by means of transgenic cotton with a reference value.

Cloning and Functional Analysis of A New Cotton Gene GhDr1 Related in Resistense To Abiotic Stresses

Abstract

Related Dissertations