| Extra short fiber cotton mutant fibers Ligonlintless (Li1) mutant and its wild type (li1) were used to study the specific genes at the elongation stage of developmental fiber by microarray. ABP and ODC were selected from eight differentially expressed genes related to fiber elongation. by bioinformation analysis for further study. The summary results were as following:1. 42 differential expression genes were obtained by analyzing the results of microarray. 8 of these genes which displayed frequently differential expression in 4 stages (-1 DPA (Day Post Anthesis), +3 DPA, +5 DPA and +7 DPA) were selected as candidate gene for further study.2. These eight target genes detected by the chip technique was verified by semi-quantitative and real-time detection, and it showed that the results by semi-quantitative PCR and quantitative real-time PCR were consistent with that of the microarray. Except that the genetic difference of individual gene was existed in one or two stages . The validity of microarray was proved.3. 42 differentially expressed genes were grouped into 12 biological pathways by KEGG (Kyoto Encyclopedia of Genes and Genomes). The highest match in the pathways involved amino acid metabolism and selenium methionine metabolism which maybe highly correlated with fiber elongation. The GO (Gene Ontology) analysis demonstrated that a single gene maybe take part in several biological process but not the only one.. All the genes could be clustered into five groups according to the differential expression level, including the down-regulated genes from-1DPA ~ +7 DPA, such as AT1G54050; the up-regulated genes from -1 DPA to +7 DPA, such as AT1G49760; the genes which were up-regulated at -1 DPA and +5 DPA, but down-regulated at +7 DPA, such as AT2G36830; and the down-regulated gene only at +3 DPA, such as AT1G12780.4. Only one base pair difference was found in the cDNA ORF (Open Read Frame) and full length DNA sequences of ABP gene in mutant Ligonlintless compared with that of wild type. which obtained by RACE . A typical C-terminal conserved domain and barrel structure which typical in ABP gene family were found by structure analysis of GhABP. Further studies showed that the genomic sequence from 1 to 52bp and from 1898 to 1948bp of GhABP in the mutant was lost which were the locations of transmembrane signal peptide binding domain. We speculated that the fiber elongation signal transduction was influenced by the mutation of this transmembrane signal gene in Li1.5. pET28a-ABP prokaryotic expression vector was constructed and induced in E. coli. Target protein band was obtained by SDS-PAGE electrophoresis analysis, the ABP protein concentration reached peak after induction for 2~4h. The success of construction of prokaryotic expression vector provided the necessary experimental data and technology to ensure the eukaryotic gene expression and functional verification of plants genetically modified in future. 6. The full length of mODC and wODC DNA senquences were obtained. The segment from 3354bp to 3530bp in this gene had been lost in the mutant, which contained many regulatory elements such as the cis-regulatory elements of the promoter, the necessary components for endosperm expression and the cis-acting regulatory elements for meristem.7. The prediction analysis of hydrophobicity, secondary structure composition, and functional areas of gene ODC revealed that there exists C-terminal and N-terminal conserved region but absent transmembrane region, ODC protein was mainly composed ofαhelix, non-lamellar structure and random coil.
|
PDF Full Text Request