| Cotton is the most important fiber crop in the world. Since cotton is usually used as raw materials of textile industry, both productivity and quality have been the main targets of cotton breeding. Although great achievement had been obtained, it was very difficulty to improve the productivity and quality of cotton fiber by traditional breeding. Gene engineering provided a new strategy and excellent perspectives to cotton breeders. Although many genes had been cloned from cotton fiber, none was directly related to the productivity and quality of cotton fiber, which have greatly prevented designing an efficient strategy to improve cotton by gene engineering.Gibberenlllins (GAs) are a class of important plant hormones, involved in various developmental processes such as seed germination, stem elongation, leaf expansion, trichome development, root growth, and the development of flower and fruit. It was demonstrated that GAs played an important role in the development of cotton fiber and related to the fiber quality. It is attractive to elucidate the relationship of GA and its biosynthesis to fiber development and fiber quality at molecular level, for which may provide a new strategy for cotton improvement.In the present research, two homologous genes of GA 20-oxidase were cloned from cotton fibers (GhGA20ox1 and GhGA20ox2). Functional analyses were performed by sequencing, bioinformatics, and overexpression in tobaccos. Finally, the functions of GhGA20ox1 gene and GAs were analyzed by overexpressing the sense and anti-sense mRNA of GhGA20oxl gene in cotton.The main results were as followings:1. Cloning of cotton GA 20-oxidase genesUsing an Arabidopsis GA 20-oxidase gene (GenBank accession No. U20872) as probe sequence, a homologous EST sequence was screened from the EST bank of cotton fiber. The corresponding genomic sequence was amplified with the special primers designed according to the EST sequence, and the 5'-upstream sequence was further amplified by a YADE (Y-shaped Adaptor Dependent Extension) method. Sequence analyses revealed that the 5'-upstream sequence contained the initiation ATG of the corresponding GA 20-oxidase gene.Using RACE method, two homologous genes of GA 20-oxidase (GhGA20ox1 and GhGA20ox2) were amplified from cotton fiber with the special primer annealing to thesequence upstream the putative ATG. The genomic sequences including the promoter sequences were further obtained by YADE method. The sequence characters and expression patterns of the two genes were analyzed by several methods such as sequence alignment, molecular hybridization and RT-PCR.The cDNA of the GhGA20oxl gene was 1401bp in length, containing an ORF of 1158bp, which encoded a polypeptide of 385 aa. Compared to the cDNA sequence, the genomic GhGA20oxl gene contained two introns of 463bp and 74bp, respectively. BLAST analysis showed that the deduced GhGA20oxl protein share high sequence similarity (53%~64% identity at amino acid level) to the GA 20-oxidases from other plant species, and also relatively low similarity to other 2-oxoglutarate dependent dioxygenases such as GA 3- P hydroxylase, GA 2-oxidase, GA 7-oxidase, flavonol synthase, flavanone 3-hydroxylase and ACC oxidase (identity ^30% at amino acid level). Multiple sequence alignment of the GhGA20oxl protein with the typical homologous proteins demonstrated that the GhGA20oxl protein contained the Fe binding site and 2-oxoglutarate binding amino acids conserved in 2-oxoglutarate dependent dioxygenases. Six conserved regions were found in the alignment of GA 20-oxidases, presumably related to the enzyme activities. RT-PCR analysis showed that the GhGA20oxl gene expressed preferentially in ovules and fibers, with the highest transcript level accumulated in the elongating fibers.The cDNA of the GhGA20ox2 gene was 1394bp in length. Sequence comparison showed that the GhGA20ox2 gene was highly homologous to the GhGA20oxl gene in the transcription region including coding regions, untranslated regions and introns, while no significant sequence similarity was found in the two promoter regions. Due to 4 deletions in the coding regions, which resulted in frame shift, the GhGA20ox2 gene could only encode two relatively short polypeptides of 115aa and 140aa, respectively. The GhGA20ox2 gene also expressed preferentially in ovules and fibers, but in more organs such as roots and the fibers at the stage of secondary cell wall thickening., compared to the GhGA20oxl gene.Using the GhGA20oxl cDNA as probe, southern analysis showed that there existed at least 3 homologous genes in the G. hirsutum genome.2. Cloning and expression profiles of the promoters of cotton GA 20-oxidase genesAccording to the 5'- upstream sequences obtained by YADE, the promoter sequences of cotton GA 20-oxidase gene GhGA20oxl and GhGA20ox2 (pGhGA20oxland pGhGA20ox2) were amplified, and the expression profile of pGhGA20ox2 was further analyzed by plant transformation.Sequence analysis revealed that the two amplified promoter sequences (pGhGA20oxl and pGhGA20ox2) were 1325bp and 1208bp, respectively. Through regulatory element searching in PlantCare web site, many regulatory elements, including those related to light response and phytohormone response were found. The expression vectors of the two promoters (pBI121-pGhGA20oxl and pBI121-pGhGA20ox2) were constructed by substituting the CaMV35S promoter in the expression vector pBI121 with pGhGA20oxl and pGhGA20ox2, respectively.The expression vector pBI121-pGhGA20ox2 was introduced into Nicotum benthamiana and cotton by Agrobacterium-medmted method, and the expression pattern of the promoter pGhGA20ox2 was determined by GUS staining. It was demonstrated that the GUS gene driven by the pGhGA20ox2 promoter specially expressed in the ovary wall and germinating seeds of N. benthamiana. In cotton, the pGhGA20ox2 promoter could direct the GUS gene to express specially in the anthers from meiosis to mononucleate stage and the elongating fibers at a relatively low level.3. Overexpression of cotton GA 20-oxidase genes in tobaccoTo construct the overexpression vector p5-GhGA20oxl and p5-GhGA20ox2, the two GA 20-oxidase genes (GhGA20oxl and GhGA20ox2) were inserted in sense into the plant expression vector p5 downstream the CaMV35S promoter.The overexpression vector p5-GhGA20oxl was introduced into N. benthamiana via Agrobacterium-mediaXed method. The overexpression of the GhGA20oxl gene generally conferred to transformants the phenotypes of GA-overproduction including elongated petioles, flowers, fruit stalks and hypocotyls of Tj seedlings, reduced leaf size, fruit size and reproductivity (seeds per fruit), and lighter green leaves. The GA4 concentrations, traced by ELISA, in the leaves of Tl plants overexpressing the GhGA20oxl gene were significantly increased compared to the untransformed plants, consistent with the elevated expression level of GhGA20oxl gene and promoted growth of plants. These observations suggested that the GhGA20oxl gene encode an active GA 20-oxidase enzyme, and promote GA biosynthesis in vivo.To compare the functions of the GhGA20oxl and GhGA20ox2 gene, common tobaccos were transformed in parallel by the two overexpression vector p5-GhGA20oxl and p5-GhGA20ox2. It was found that over-expression of the GhGA20oxl gene also conferred the phenotypes of GA-overproduction to transformed common tobacco,while the GhGA20ox2 gene had no significant effect on the growth of transformants, suggesting that the GhGA20ox2 gene could not encode any active GA 20-oxidase enzyme. The function(s) of the GhGA20ox2 gene in cotton is still to be elucidated.4. Overexpression and anti-sense suppression of GhGA20oxl gene in cottonThe anti-sense suppression vector of the GhGA20oxl gene (p5-anti-GhGA20oxl) was constructed by inserting the GhGA20oxl gene at reverse direction into the expression vector p5 downstream the CaMV35S promoter.Cotton was transformed in parallel with the expression vectors p5-GhGA20oxl, p5-anti-GhGA20oxl and p5. Thirteen GhGA20oxl overexpressing plants, 18 anti-sense GhGA20oxl plants and 2 p5-transformed plants were obtained. Northern blotting and RT-PCR analysis indicated that both the sense and anti-sense GhGA20oxl gene could express in the transgenic cottons.The altered phenotypes in the transgenic cottons suggested that the overexpresion and anti-sense suppression of the GhGA20oxl gene influenced many aspects of cotton development, including growth of stems and leaves, floral development, the development of seed and fibers, suggesting that GA were involved in these developmental processes.Compared to the control (p5-transformed plant), the stems of the GhGA20oxl overexpression plants elongated faster and had longer internodes, while the stems of anti-sense GhGA20oxl plants elongated slower and had shorter internodes. Spraying GA3 could restore the growth of the stems of anti-sense suppression plants. These observations indicated that the variation of stem growth in the transgenic cottons could mainly attribute to changes in GA biosynthesis, which is related to the overexpresion and anti-sense suppression of the GhGA20oxl gene.Compared to the control and the GhGA20oxl overexpression plants, the duration of floral development of the anti-sense GhGA20oxl plants were significantly longer, and the flowers were totally male sterile, indicating that GAs were ivolved in floral development of cotton. Histological section revealed that the dissociation of tetraspores and the disintegration of tapetum in the course of anther development of the anti-sense GhGA20oxl plants were hindered. To our knowledge, this is the first observation indicating that GA may be involved in the dissociation of tetraspores and the disintegration of tapetum.Though pollinated, the boll development of the anti-sense GhGA20oxl plants... |
PDF Full Text Request