| Gossypium, a fiber and oilseed crop, played an important role in the econmy and commodity worldwide. Botanically, the cotton fibers, special cell with less cell inclusions but more cell wall, developing from individual epidermal cell on the ovules of cotton, were unicellular. The sea island cotton(Gossypium babardense L.) has been highly valued in verticillium wilt resistance and many fiber qualities including fiber length, strength and fineness. To identify whether it had some special genes in fiber development in comparison with the upland cotton(G. hirsutum L.), homogalacturonans transferase(GAUT) gene and an actin-deploymerizing factor(ADF) gene were cloned and characterized in this research. The fiber development was very similar to plant cell wall morphogenesis. The genes of glycosyltransferases 8 family, such as pectic, xylans and xlyoglucans polysaccharides, had been revealed coexpressing with cellulose enzymes in Arabidopsis and Aspen, and maybe played an important role in cotton fiber development. Homogalacturonans transferase(GAUT) gene was cloned from Pima 90-53(G. babardense L.) with homologybased cloning, which related with fiber strength. And an actin-deploymerizing factor(ADF) gene, which highly conserved in all eukaryotic cells, played a key role in the cell elongation, cell division and so on. The research attempted to obtain the full length of these two cDNA fragments by in silico cloning, RT-PCR and RACE. And the functions of the two genes were studied preliminarily. The main results were as follows:1 Three partial gDNA fragments of GbGAUT1, GbGAUT2 and GbGAUT3 genes were amplified by several degenerate and special primers pairs. Comparing the three fragments with AtGAUT1, there were about homologous 52%(55/104), 51%(57/99) and 51%(54/104), respectively. All of them had partial GT8 family conserved domain.2 Full length cDNA of GbGAUT1 was cloned by in silico cloning, RT-PCR and RACE, which was 2528 bp, including a full length ORF of 1797 bp, a 269 bp 5′-UTR and a 462 bp 3′-UTR, and concluding a full length GT8 family conserved domain. The deduced protein was predicated to contain 598 amino acid residues, which was classified stable protein with a mass of 45.1% α-helixes and 34.1% β-sheet. A theoretical molecular weight of GbGAUT1 was 69.46 kDa and a theoretical pI was 8.16. GbGAUT1 contained several N-glycosylation sites, several phosphorylation sites and N-myristoylation site by bioinformation analysis on line. DxD, a conserved domain of GT-A superfamily, was found in GbGAUT1 and a transferase stack coiled coil glycoprotein signal-anchor expressed Golgi was scaned by PROSITE database in NPS online. A calmodulin combine region was found by CAM binding site search and analysis. GbGAUT1 could be considered as a homogalacturonan(HG) transferase protein since the conserved GAUT1-related superfamily motif [H-[FWY]-[DNS]-G-x(2)K-P-W-x(2)-[ILM]-[ADGS] had been found. Semiquantitative RT-PCR showed that the expression levels of GbGAUT1 in 10 DPA and 35 DPA fiber were higher than other tissues and other developmental period of cotton fiber, and especially in 35 DPA.3 An 813 bp gDNA of GhADF1 were obtained by amplified, which contained a 420 bp full length ORF cDNA. There was about homologous 99% with GhADF1 and GhADF8. An 84 bp intron near the 3′ end at the beginning of the PIP2/actin binding site(Trp 90) from 287 bp to 371 bp in ORF of gDNA. The deduced protein was predicated to contain 139 amino acid residues, which was classified stable protein with a mass of 66.9% α-helixes and 22.3% β-sheet. A theoretical pI was 5.47. A calmodulin combine region was found by CAM binding site search and analysis. Hydropathy and transmenber motif analysis indicated that GbADF1 was a nontrans-membrane hydrophobe protein. GbADF1 contained a conserved Ser(A) phosphorylation sites in N-terminus and a conserved PIP2/actin binding site by bioinformation analysis on line. Semiquantitative RT-PCR showed that GbADF1 was a constitutive expression gene in cotton; somewhat higher levels were detected in fibers than in trophic tissues.4 To characterize the GbGAUT1, the cDNA was expressed in E. coli. A pair of primers was designed in the light of the start codon and stop codon sites, the full length ORF was amplified by using cDNA template. At the same time, the ORF without signal was amplifyied by RT-PCR. The prokaryotic expression vector pET-GbGAUT1 was constructed directe. Compared to the control of non plasmid E. coli cell and harboring pET-41a-c(+), the E. coli cell harboring pET-GbGAUT1 could be induced by IPTG, and the recombinant GST-tagged GbGAUT1 without signal was expressed. After induction, the recombinant GbGAUT1 was calculated by SDS-PAGE, approximately 92 k Da. The protein before the multiple cloning sites, including GST was about 31 kDa in the vector. Then GbGAUT1 was a 61 kDa peptide.5 So far, no cotton ADFs were expressed in E. coli. To characterize the GbADF1, the cDNA was expressed in E. coli. A pair of primers was designed in the light of the start codon and stop codon sites, the full length ORF was amplified by using cDNA template. The prokaryotic expression vector pET-GbADF1 was constructed by middle-recombine plasmid pMD-GbADF1. Compared to the control of non plasmid E. coli cell and harboring pET-41a-c(+), the E. coli cell harboring pET-GbADF1 could be induced by IPTG, and the recombinant GST-tagged GbADF1 was expressed. After induction, the recombinant GbADF1 was calculated by SDS-PAGE, approximately 50 kDa. The protein before the multiple cloning sites, including GST was about 31 kDa in the vector. Then GbADF1 was a 19 kDa peptide.6 The plant translation vector of GbGAUT1 and GbADF1 were built and the full length ORF was amplified by RT-PCR, respectively. The plant expression vector pGN-GbADF1 and pGN-GbGAUT1 were constructed by middle-recombine plasmid, and the results were testified by two enzyme cutting, PCR amplification and sequencing.7 To widen the genotype of regeneration cotton, Nongda-94-7 was studied, which was an upland cotton variety crossing from many elite lines in Agroculture university of Hebei China. It had well agronomic properties and highly anti-disease characters, not having been studied on somatic embryogenesis. The success of regeneration in Nongda 94-7 made a good passway to breed transgenic cottons with independent intellectual property.8 Cotton, tobacco and Arabidopsis were transformed overexpression vector pGN-GbADF1 and pGN-GbGAUT1 via Agrobacterium-mediated transformation. Using the method of floral dip, overexpression vector pGN-GbGAUT1 was transferred into homologyical mutation, irx8 of Arabidopsis Columbia and wild. |
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