| Cotton fibre is a widely used raw material in textile industry. It plays an important role in national economy and people’s lives. It is a tubular single cell derived from the outer epidermis of ovule. And it is an ideal mode material for the investigation of polar elongation development of plant cell. So, it is important to elucidate fiber development process and major factors related to fiber elongation development by molecular biology. In this study, we applied the microarray, cDNA library screening and cotton transformation technology to isolate and characterize genes related to cotton fiber development. This would help us to understand the mechanism of polar elongation development of fiber cell. The mainly result composed of three sections:1) ESTs analysis during early fiber elongation development stagesA cDNA library was constructed using 7235 fibers at 5 to 25 DPA. At this time, the fiber cells were in the process of elongation and synthesis of secondary cell wall cellulose. Random sequencing generated 1436 ESTs and a microarray containing the 1436 ESTs was subsequently identified by reciprocal hybridization using fiber mRNAs of upland cotton wild type XZ142 and its lintless-fuzzless mutant fl.152 up-regulated ESTs were obtained, including 41 up-regulated ESTs in 2DPA,4DPA and 6DPA; 4 up-regulated ESTs in 2DPA and 4DPA; 14 up-regulated ESTs in 4DPA and 6DPA; 12 up-regulated ESTs in 2DPA and 6DPA; 12,15 and 54 up-regulated ESTs in 2DPA,4DPA and 6DPA, respectively.65 down-regulated ESTs were obtained, including 1 down-regulated EST in 2DPA,4DPA and 6DPA; 10 down-regulated ESTs in 4DPA and 6DPA; 1 down-regulated EST in 2DPA and 6DPA; 1,14 and 38 down-regulated ESTs in 2DPA,4DPA and 6DPA, respectively. The functional category of differentially expressed ESTs included energy/carbohydrate metabolism (ECM), cellular structure and organization (CSO), protein metabolism, transport, lipid metabolism, cytoskeleton, transcription, environmental response and defense (ERD), nucleic acid metabolism, signaling, hormone, secondary mechanism, and the ESTs belonged to ECM were the most. In order to further confirm the data from the microarray analysis, RT-PCR was performed for the differentially expressed ESTs.73 pairs of primers were designed from 217 differentially expressed ESTs. And the RT-PCR results were produced from 34 of 73 pairs of primers. The results indicated that 24 pairs of primers were coincident with the microarray results and the positive proportion was 70.6%. Using RT-PCR primers as markers for chromosome mapping,26 polymorphisms loci produced by 19 ESTs RT-PCR primers were integrated into our backbone map. This study provided a platform for isolating and characterizing genes related to cotton fiber primary development.2) Cloning and characterization of fiber annexin genes GhFAnnl and GhFAnnxTwo clones encoded fiber annexin protein, were separated from developmentally different cotton fiber library constructed from super-fiber quality line 7235, designated GhFAnnl and GhFAnnx. The insert fragment of GhFAnnl cDNA clone is 1200bp, and two fragments of 724bp and 747bp of 5’upstream were obtained via TAIL-PCR technique. After integrated with the original sequence, we acquire a cDNA sequence accounting for 1800bp. Its open reading frame is 951bp, and encodes a polypeptide containing 316 amino acids, and the Mw of the deduced amino acid is 36.0kDa, and pI is predicted to be 6.34. There exists an in-frame stop codon in the upstream. Blast analysis indicates that it had a 98 % identity with an annexin gene FAnn reported in cotton at nucleic acid level of the ORF region. Judging from its expression characters, GhFAnnl is highly expressed in roots, stems, leaves and fiber cells of different stages, but it is faintly decreased at 17DPA of fiber cells. Southern blotting analysis shows that there are two copies of GhFAnnl in the genome of upland cotton; sub-genome A and sub-genome D contains each. Genomic GhFAnnl sequences were subsequently isolated from different cotton species, TM-1, Hai7124 and two diploid progenitor cottons, G. herbaceum (A-genome) and G. raimondii (D-genome). And the result indicated that GhFAnnl came from A-genome. GhFAnnl was mapped on chromosome D5. The insert of fragment of GhFAnnx cDNA clone is 1157bp. Its open reading frame is 945bp, and encodes a polypeptide containing 314 amino acids, and the Mw of the deduced amino acid is 35.7kDa, and pI is predicted to be 6.49. There exists an in-frame stop codon in the upstream. Blast analysis indicates that it shares a high homological identity with an annexin gene from strawberry, and it is a new member of annexins family of cotton fiber. Judging from its expression characters, the level of GhFAnnx transcripts is low in roots, stems and leaves, and abundant in all the developmental stages of the fiber cells and slightly reduced at 17DPA. Northern blotting analysis confirms that the transcripts of GhFAnnx in roots, stems and leaves is low and is highly expressed at 5DPA,8DPA,11DPA and 14DPA of fiber cells, and is suddenly reduced at 17DPA. Genomic GhFAnnx sequences were subsequently isolated from different cotton species, TM-1, Hai7124 and two diploid progenitor cottons, G. herbaceum (A-genome) and G. raimondii (D-genome). And one of GhFAnnx copies was mapped on chromosome A10.3) Preliminary transgenic functional analysis of two annexin genesTo make clear the function of GhFAnnl and GhFAnnx, we constructed a series of different expression vectors, and transformed into plant cells via different transformation methods to analyze the function of annexins gene in plant cells. First, we constructed two vectors fused with GFP report gene, and transformed into the epidermal cells of onion and ODPA ovules of cotton via gene gun to analyze the subcellular localization of two genes in plant cells. After transformation of the epidermal cells of onion via gene gun, the information of localization results of two genes were similar, and the results indicated that the green fluorescence was ubiquitous in the epidermal cells of onion. And the results indicated that the GFP was assembled in cellular membrane and not in the cellular wall by treatment of 20%sucrose. Further more, after transformation of ODPA ovules of cotton via gene gun, the fiber were peeled off to be observed when ODPA ovules cultured 14DPA later in vitro. The results indicated that the localization results of two genes were all similar and the GFP were assembled at the edge and on the inner side of the apex of the cotton fiber tip with brilliant spots. Second, we constructed 6 expression vectors of GhFAnnl and GhFAnnx, including sense expression vector of GhFAnnl fused with GUS gene (SAnn1), anti-sense expression vector of full length of GhFAnnl gene (ASAnn1), anti-sense expression vector of 5’-upstream fragment of GhFAnnl gene (3ASAnn1), sense expression vector of GhFAnnx fused with GUS gene (SAnn2), anti-sense expression vector of full length of GhFAnnx gene (ASAnn2), interfered expressed vector (IAnn2). Then, all 6 plant-expressed vectors were transformed into the cotton genome by the Agrobacterium-mediated transformation. All of transgenic plants were detected by the PCR analysis using the NPTII and promoter-gene specific primers. And the histochemistry analysis of SAnnl and SAnn2 were employed to detect the expression localization characterization of GhFAnnl and GhFAnnx. According to the GUS dyeing of cotton embryogenic calli from Agrobacterium-mediated transformation, the result indicates that the expression pattern of reported gene GUS is chimeric. In addition, the histochemistry analysis of rhizoid of positive SAnnl and SAnn2 transgenic plants were employed to study the association between annexins and cell secretion. It indicated that annexins were assembled at the tip of the rhizoid. Different vectors of transgenic plants of GhFAnnl and GhFAnnx were obtained, and it provided a platform for further functions analysis of annexins gene in the polar development and physiological processes of cotton fiber. |
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