| Cotton is one of the most important economic crops in the world, which supplies natural fiber for textile industry. Cotton fiber initiates from ovule epidermis cells and its development consists of four overlapping stages: fiber initiation, cell elongation, secondary wall deposition and maturation. According to the"ABCDE"model of flower development, AG-lineages are essential to both ovule and ovary development, which are determinate factors for fiber yield. The development of cotton fiber is a complicated process involving the strict regulation of a large amount of genes. Though its mechanism is still unclear, some transcription factors related to fiber development have been isolated from cotton, among which the MYB transcription factors are very attractive for researchers to elucidate the fiber developmental mechanism. As AG-lineages and MYB transcription factors play crucial roles in cotton fiber development, cloning and analysis of these two kinds of transcription factors might provide useful candidate genes for molecular breeding in improving cotton yield and quality.In this study, two full-length cDNAs of AG-lineages, designated as GbAGL1 (Genbank accession FJ198049) and GbAGL2 (Genbank accession FJ198050) and three full-length cDNAs of MYB proteins, designated as GbMYB1 (Genbank accession FJ198051), GbMYB2 (Genbank accession FJ198052) and GbMYB3 (Genbank accession FJ198053) were isolated by RACE from +3 DPA ovule tissues of Gossypium barbadense for the first time. Subsequently, Southern blot, RT-PCR, RNA in situ hybridization, Real-time PCR and transgenic Arabidopsis using these genes were used to study these genes'features and expression patterns.The full-length cDNA of GbAGL1 consisted of 951 bp with a 67 bp 5'UTR, a 212 bp 3'UTR and a 672 bp ORF, which encoded a polypeptide of 223 amino acids (protein accession: ACI23560) with a calculated molecular weight of 25.72 kDa and pI of 9.44. The deduced GbAGL1 protein contained a MADS-box from residues 2 to 56, a less conserved K-box from residues 91 to 157, and two AG motifs (AG motif I from residues 194 to 206 and AG motif II from residues 211 to 223). GbAGL1 exhibited high similarity with other members of D-lineages, suggesting that GbAGL1 belongs to D-lineages and may be involved in the ovule development in cotton. Genomic DNA sequence analysis and Southern blot analysis indicated that GbAGL1 belonged to a low-copy gene family in G. barbadense. RT-PCR analysis showed that GbAGL1 expressed highly in ovules but low in roots, stems and leaves. The transcriptional signals were gradually and regularly intensified from -3 DPA to +8 DPA. In situ hybridization analysis of GbAGL1 in flower buds and ovules at different stages indicated that GbAGL1 mRNA was expressed in the whole floral bud primodia and floral organs including ovules and fibers. Over-expression of GbAGL1 in transgenic arabidopsis caused homeotic alternations in floral organs, which were the typical phenotypes of other D-lineages. These results indicate that GbAGL1 is involved in ovule development and might play roles in the development of cotton fiber.The full-length cDNA of GbAGL2 consisted of 1098 bp with a 110 bp 5'UTR, a 252 bp 3'UTR and a 735 bp ORF, which encoded a polypeptide of 244 amino acids (protein accession: ACI23561) with a calculated molecular weight of 28.12 kDa and pI of 9.34. The deduced GbAGL2 protein contained a MADS-box from residues17 to 71, a K-box from residues 105 to 171, and two AG motifs (AG motif I from residues 214 to 226 and AG motif II from residues 232 to 244). GbAGL2 shared high similarity with other members of C-lineages, suggesting that GbAGL2 belongs to C-lineages and may be involved in the ovary development in cotton plants. Genomic DNA sequence analysis and Southern blot analysis indicated that GbAGL2 belonged to a low-copy gene family in G. barbadense. RT-PCR analysis showed that GbAGL2 expressed highly in ovules and fiber tissues but low in roots, stems and leaves. In situ hybridization analysis of GbAGL2 in flower buds and ovules of different developmental stages indicated that GbAGL2 mRNA was expressed in the ovary primodia, ovules and fibers. Over-expression of GbAGL2 in transgenic Arabidopsis caused homeotic alternations in floral organs, which were the typical phenotypes of other C-lineages. These results indicated that GbAGL2 was involved in ovary development and might play roles in fiber development.The full-length of GbMYB1 consisted of 974 bp with a 66 bp 5'UTR, a 143 bp 3'UTR and a 765 bp ORF, which encoded a polypeptide of 254 amino acids (protein accession: ACI23562) with a calculated molecular weight of 29.07 kDa and pI of 6.25. The deduced GbMYB1 protein contained two MYB repeats from residues 14 to 61 and 67 to 112. GbMYB1 shared high similarity with other members of proteins, suggesting that GbMYB1 gene belongs to the R2R3-MYB type transcription factors and may be involved in fiber development in G. barbadense. Genomic DNA sequence analysis and Southern blot analysis indicated that GbMYB1 belonged to a low-copy gene family in G. barbadense. RT-PCR analysis showed that GbMYB1 expressed highly in ovules but low in roots, stems and leaves. The transcriptional signals were gradually and regularly intensified from -3 DPA to +8 DPA. The results of Real-time PCR analysis of GbMYB1 expression in ovules of different developmental stages were in accordance with that of RT-PCR data. Furthermore, the GbMYB1's expression (ΔCT value) in XZ142 (wt) exceeded that in the fibreless mutant XZ142 (fl). In situ hybridization analysis of GbMYB1 in ovules at different stages indicated that GbMYB1 mRNA was accumulated in ovule outer layers and fibers. Over-expression of GbMYB1 in transgenic Arabidopsis caused abnormal phenotypes including narrower leaves, lower plant height, shorter roots and shorter siliques.The full-length of GbMYB2 consisted of 979 bp with a 163 bp 5'UTR, a 72 bp 3'UTR and a 747 bp ORF, which encoded a polypeptide of 248 amino acids (protein accession: ACI23563) with a calculated molecular weight of 28.09 kDa and pI of 8.99. The deduced GbMYB2 protein contained two MYB repeats from residues 25 to 72 and 78 to 123. GbMYB2 shared high similarity with other members of R2R3-MYB proteins, suggesting that GbMYB2 belongs to the R2R3-MYB type transcription factors in G. barbadense. Genomic DNA sequence analysis and Southern blot analysis indicated that GbMYB2 belonged to a low-copy gene family in G. barbadense. RT-PCR analysis showed that GbMYB2 expressed in ovules and other tissues. Real-time PCR analysis indicated that from -3 DPA to +12 DPA, the GbMYB2's expression (ΔCT value) in ovules of different developmental stages was stronger regularly. Furthermore, the GbMYB2's expression (ΔCT value) in XZ142 (wt) exceeded that in the fibreless mutant XZ142 (fl). In situ hybridization analysis of GbMYB2 in ovules at different stages indicated that GbMYB2 mRNA was accumulated in ovule outer layers and fibers. Over-expression of GbMYB2 in transgenic Arabidopsis caused abnormal phenotypes including broaden and wrinkle leaves, more dense leaf trichomes, longer roots, delayed flowering stage, and increased silique length. These results indicated that GbMYB2 is involved in leaf trichome development and maybe play roles in fiber development.The full-length of GbMYB3 consisted of 1129 bp with a 73 bp 5'UTR and a 271 bp 3'UTR and a 795 bp ORF, which encoded a polypeptide of 264 amino acids (protein accession: ACI23564) with a calculated molecular weight of 29.63 kDa and pI of 9.12. The deduced GbMYB3 protein contained two MYB repeats from residues 14 to 61 and 67 to 112. GbMYB3 shared high similarity with R2R3-MYB type proteins, suggesting that GbMYB1 belongs to the R2R3-MYB type transcription factors and may be involved in fiber development in G. barbadense. Genomic DNA sequence analysis and Southern blot analysis indicated that GbMYB3 belonged to a low-copy gene family in G. barbadense. RT-PCR analysis showed that GbMYB3 expressed in ovules and other tissues. Real-time PCR analysis indicated that the GbMYB3's expression (ΔCT value) in ovules of different developmental stages was gradually and regularly intensified from -3 DPA to +12 DPA. Furthermore, the GbMYB3's expression (ΔCT value) in XZ142 (wt) exceeded that in the fibreless mutant XZ142 (fl) appreciably. In situ hybridization analysis of GbMYB3 in ovules at different developmental stages indicated GbMYB3 mRNA was accumulated in ovule outer layers and fibers. Over-expression of GbMYB3 in transgenic Arabidopsis caused abnormal phenotypes including shrinkage leaves, short roots, and short siliques. |
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