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Cloning and Characterization of Two Genes Related with Fiber Development and Expression Analysis of Seven Genes Related with Fatty Acid Metabolism in Cotton

Author: DongJia
Tutor: GuoWangZhen
School: Nanjing Agricultural College
Course: Genetics
Keywords: GhEG GhGLU fatty acid metabolism structure character expression analysis stress
CLC: S562
Type: Master's thesis
Year: 2010
Downloads: 8
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Abstract


Cotton is not only useful fiber plant, but also important oil crops. Although molecular biology research in cotton was fast, the rsearch has mainly focused on improving the fiber yield and quality. Correspondingly, the research on improving the cotton seed oil and resisting of the abiotic stress in cotton by genetic engineering of fatty acid matabolism have not been regarded until now. Cloning and expressional analysis of genes related both with fiber development and the cotton seedoil were carried out in the study, the results as follows:1 Molecular cloning and chatacterization of two genes(GhEG and GhGLU) related with fiber development in Gossipium.Based on the difference of expression of EST on genechip with the materials of upland cotton standard line TM-1 and the sea-island cotton cultivar Hai7124, the gene of full-lenghth sequence was obtained by sillico cloning. And sequence analyses showed that the size of cDNA was 2188bp, and it included an open reading frame of 1581bp that encoded a polypeptide of 526 amino acids and the protein weight was 22.1KDa and a calculated pI was 5.90. The further research isolated the gene genomic sequence from TM-1, Hai7124, G. raimondii and G. herbaceum. The result of Q-PCR indicated that this gene is expressed mainly in fiber elongation and have obviousely difference between the two materials of TM-1 and Hai7124, which is identical with the result from genechip. By comparing the genomic sequence between TM-1 and Hai7124, the marks of SNAP was developed. Further using the BC1 mapping population derived from the hybridization between upland cotton standard line TM-1 and the sea-island cotton cultivar Hai7124, GhEG was localized on chromosome 19. By screening cDNA library of G. hirsutum acc 7235, a novel cotton gene, named GhGLU, was cloned. The full lenghth of this gene was 2088bp, ORF was 1410bp, encoding 469 aa. The protein molecular weight was 50.30292kDa and a calculated pI was 5.307. The GhGLU express preferentially in root, fiber initiation and elongation and have obviouse difference between TM-1 and Hai7124. By comparing the genomic sequence between TM-1 and Hai7124, the marker of SNAP was developed. Further, using the BC1 mapping population derived from the hybridization between upland cotton standard line TM-1 and the sea-island cotton cultivar Hai7124, GhEG was localized on chromosome 4.2 Expression analyses of seven genes related with fatty acid metabolism in cottonBased on the full-lenghth cDNA of fatty acid metabolism related genes, we first analyses these genes by the expression of tissues and induced-expression in cotton. Tissue expression pattern analysis revealed that these genes expressed in all the tested tissues at different developing stages. The transcripts of these genes in leaves were higher than that in root and stem. In different fiber developmental stages, GhKASII, GhKASIII showed the highest expression level in seeds at 25 DPA, however, GhGPAT have the strongest expression level in ovules at 0 DPA. The GhFADS gene transcript was the highest in fibers at 20 DPA. GhACX showed the highest expression level in seeds at 25 DPA, however, GhMFP and Gh4CL have the strongest expression level in ovules at 0 DPA. These results inferred that some genes not only participate in fiber development, but also contribute to metabolism of fatty acids, in detail, the cotton seed expanded during the fiber development, and fatty acids also synthesized. In addition, based on real-time quantitative RT-PCR, these genes were differentially regulated under wounding, methyl jasmonate (Meja), cold and ABA (abscisic acid) conditions. These results indicated that these genes may play important in resistiance to stress stimuli.

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