Cloning And Functional Characterization Of GhDUF231L1Gene In Cotton(Gossypium Hirsutum)

Cotton(Gossypium hirsutum) is one of the world’s most important fiber crops and cash crops, so improving the quality of the cotton fibers has significant meaning. During cotton breeding, simply control environmental factors to improve cotton production can’t satisfy people’s needs now. Using biochemical and molecular biology techniques to improve the quality of the cotton appears especially important. The genes of DUF231family encode the protein with domain of unknown function231and there is not much research on this gene family currently. Functions of this gene family have been found to be associated with the pectin and hemicellulose of the cell wall in Arabidopsis. In this thesis, our studies are mainly focused on the function of GhDUF231L1. The main results are as follows:1. Cloning and characterization of GhDUF231L1We got the predicted sequence of GhDUF231Ll gene by electronic cloning. Sequence analysis of GhDUF231L1pritein showed that GhDUF231L1gene had five exons and four introns. And the open reading frame (ORF) of GhDUF231L1was1293bp in length and encoded a protein of430amino acids. The further analysis result shown that GhDUF231L1contained a transmembrane domain structure in the N-terminal domain and an unknown function structure in the C-terminal domain.2. Expression of GhDUF231L1in cotton tissuesWe analyzed the tissue expression of GhDUF231L1by RT-PCR. The result revealed that GhDUF231L1was preferentially expressed in cotton fiber. Its expression gradually increased during the deposition stage of fiber secondary cell wall(15-20DPA).The expression level of GhDUF231L1was low in tissues such as roots and hypocotyls, and almost had no expression in other tissues. This indicated that GhDUF231L1had function in regulating the development of cotton fibers seconderay cell wall.3. Analysis of GhDUF231L1promoter activityTo analysis the activity of GhDUF231L1promoter, we cloned the sequence of GhDUF231L1promoter. A construct in which GUS was placed under the control of the GhDUF231L1promoter were transformed into Arabidopsis, and we gained several transgenic lines. The result of GUS staining showed that GUS signals were found in rosette leaves (leaf vein), roots, stem (vascular tissue), floral tissues and stliques.The sequences of different lengths which belonged to GhDUF231L1promoter sequence fused with the GUS gene and introduced into Arabidopsis plants. Then we got the stem cross-section at10cm above rosctte-level when the plants were six weeks old, and stained them. GUS staining resulting from GhDUF231L1promoter activity was found strongly in the xylem cells of the stem; GUS activity driven by GhDUF231L1promoter without SNBE1sequence was localized to the cortex and xylem vessel; GUS activity driven by GhDUF231L1promoter without SNBE2was detected in cortex and phloem; GUS staining resulting from GhDUF231L1promoter without SNBE1/2activity was found in cortex and phloem, but the signal was stronger. These results indicated that SNBE may be important for the function of GhDUF231LI promoter.4. Subcellular localization of GhDUF231L1proteinGhDUF231L1was predicted to be a membrane protein by TMHMM analysis software. To reveal the intracellular localization of GhDUF231L1protein, an eGFP reporter gene was fused to GhDUF231L1. This GhDUF231L1-eGFP construct was introduced into tobacco(Nicotiana tabacum) epidermal cells by agroinfiltration. After a subculture of48hours, the GFP fluorescence was observed by confocal microscopy. The GFP fluorescence signals were detected in cytomembrane of the transformed tobacco cells, suggesting that GhDUF231L1protein may localize in the cytomembrane of cells to perform its function in cotton.5. Phenotypic analysis of GhDUF231L1overexpression transgenic ArabidopsisIn order to study the function of GhDUF231L1, We got the tbl3mutants from professor Wolf-Rudiger Scheible. PCR analysis on gDNA and cDNA levels showed tbl3mutants were homozygous lines. Measurement of stem cellulose content showed that the tbl3mutants had slight but significant reductions of crystalline cellulose as compared with WT. More specifically, the rosette leaves of the tbl3mutants showed irregular curly compared to WT. To gain further insight into the leaves phenotype, section analysis of petioles was performed with tbl3and WT. The arrangement of vascular cell was disorderly in tbl3. This may be the reason which led to the distinguishable changes of tbl3phenotype.We constructed the over-expressing GhDUF231L1vector. And then we transferred the recombinant vector into Arabidopsis. After getting the GhDUF231overexpression transgenic plants, we detected the phenotypes of six-week old transgenic plants, tbl3mutants and WT. GhDUF231L1overexpresion transgenic plants were highest, and tbl3mutants were the shortest. We also analyzed the cellulose content of WT and transgenic lines. Compared with WT, all the transgenic lines contained relatively high levels of cellulose. This indicated that overexpression of GhDUF231L1in wild type Arabidopsis increased cellulose content.To further validate whether GhDUF231L1affected the cell wall components, we isolate monosaccharide compositions of WT, tbl3and GhDUF231L1overexpression transgenic plants from stems. The analysis of gas chromatography shown that the content of monosaccharide compositions in GhDUF231L1overexpresion plants had changed.