| Cotton (Gossypium spp.) is an important economic crop and the largest source of textile fiber in the world. Cotton fibers used in textiles are derived from the pubescence of maturing seeds. The high fiber quality is one of the major goals in the cotton breeding all over the world. So, it is important to elucidate fiber development process and major factors related to fiber quality by molecular biology. In this study,+1DPA, ODPA,-1 DPA ovule from isogenic lines Xinxiangxiaojilintless-fuzzless mutants (XinWX) and Xinxiangxiaojilinted-fuzzless mutants (XinFLM) were used as the material, using cDNA microarray to select the genes related to the initiation of cotton fiber and preliminary analysis the roles of the genes in physiological and biochemical processes of cotton;Then, the molecular cloning of Ghhep, GhAOC and GhGDSL was processed. The expression of Ghhep and GhAOC showed that they were related with secondary wall thickening during the development in cotton, and the expression of GhGDSL was related with the fiber elongation during the development in cotton.(1) The XinWX, XinFLM early developmental stages (+1DPA, ODPA,-1DPA) of ovule as the experimental material for competitive microarray analysis (hereinafter referred to as hybrid chip +1DPA, ODPA,-1DPA), Comparative analysis of transcriptomes by cDNA microarray showed 1948 genes were differentially expressed at the fibre initiation developmental stages. Of these,874 genes were differentially expressed at +1DPA,325 genes were differentially expressed at ODPA,749 genes were differentially expressed at-1DPA.(2) Analyses of Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed genes indicated biological progress primarily involved in binding activity:391 genes, catalytic activity:383 genes, transporter activity:59 genes, transcription regulator activity:38 genes, molecular transducer activity:22 genes, structural molecule activity:13 genes, enzyme regulator activity:9 genes, motor activity:5 genes, antioxidant activity:3 genes, nutrient reservoir activity:2 genes, translation regulator activity:2 genes, other function:6 genes. The 383 genes related with catalytic activity were further classified into 9 subgroups: transferase activity:131 genes, hydrolase activity:107 genes, oxidoreductase activity:63 genes, lyase activity:34 genes, ligase activity:11 genes, isomerase activity:7 genes, deaminase activity:1 gene, glycogen debranching enzyme activity:1 gene, UDP-L-rhamnose synthase activity:1 gene, other function:57 genes.(3) Using quantitative RT-PCR (Q-PCR) analysis, we selected the 120 differentially expressed genes to confirm the realitabilty of chip hybridization results, the selected genes involving oxidoreductase activity, hydrolase activity, binding activity, and transport activity. Of them, the developed primer pairs of 60 differentially expressed genes can effectively amplify the PCR product, while another 60 not. Further,15 had not significant difference in fiber intiation stages between two parents, and 45 showed the significant difference which was consistent with the chip hybridization results. The positive rate was 75%. The results showed that there are some false positive in the chip, but most of gene expression results are consistent with the chip.(4) Based on chip verification, two categories of differentially expressed genes: hydrolase activity and oxidoreductase activity have been further analyzed the expression characterization in different tissues and organs using cotton genetic standard line TM-1. The results showed different genes had different preferential expressional tissues or organs. Three genes, encoding adenosine 5'phosphosulfate reductase,GDSL-motif lipase/hydrolase protein and endo-1,4-beta-glucanase, were related with fiber elongation development;Two genes, encoding pullulanase (starch debranching enzyme) and non-intrinsic ABC protein were related with secondary wall thickening;Three genes, encoding ABC-transporter-like protein,xyloglucan endotransgly- cosylase and expressed protein were related with initial phase of fiber development;Three genes, encoding xyloglucan endotransglycosylase,protein phosphatase 2C homolog and pherophorin-C1 protein precursor were related with seeds development;Three genes, encoding peroxidase,mitochondrial alternative oxidase 2 and metallopeptidase were related with root, stem and leaf development. (5) Using homologous sequence alignment and splicing EST online-cloning, three full length cDNA related with fiber development, named Ghhep, GhAOC and GhGDSL, were cloned. Using quantitative RT-PCR (Q-PCR) analysis, the expression of Ghhep and GhAOC were related with the secondary wall thickening, and the expression of GhGDSL was related with the fiber elongation. |
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