| The planting pattern of "dwarf plant,high plant dense and early maturation" is the major cotton planting technique in Xinjiang province of China, which have led to a rapid increase in yield per unit area. Studying on cotton plant height, especially, identification of genetic resources controlled plant height has been becoming one of the most important tasks for the cotton production. The financial burden on farmers increased for the additional aplication of mepiquat chloride every year. Therefore, it is a target for breeders to select the dwarf cotton varieties with excellent fiber quality. A novel cotton dwarf mutant AS98 with extremely short stems but normal leaf shape was discovered in a F2 population of interspecific hybridization in 1998. Compared with the other dwarf cottons with crinkled leaves that had been reported previously, the leaves of it were normal shape. After self pollination for several generations, homozygous mutant have been gained. The mutant crossed with wild-type could produce the short (about 50 cm) and compact plants which had little difference in fiber qualities compared with that of the wild-type. In this study, based on the cell and tissue characters of the dwarf mutant, the related genes and regulation element of dwarf mutant was identified and analyzed. This will lay a foundation for genetic improvement and effiently utilizing this dwarf mutant. To this end, the main results obtained are as follows:1. Cells and histological features of the dwarf mutant. We found that the enlarge-growth of mutant cells was severely affected by comparison and analysis of different characteristics of cells and tissues of the normal strain and dwarf material through the microscope. Through electron microscopy we found that the cell wall of most dwarf cells was thickening. After treatment with exogenous GA3, the cells can increase with the cell wall thinning. In the contrary, the cells of normal plants were restrained with thicker cell walls after treatment of paclobutrazol. This demonstrated that the growth of the plant stem was tied to thickness of cell walls.2. Three Expansin genes associated with cell wall expansion of the dwarf mutant were identified and screened by RT-PCR and QRT-PCR techniques. The expansion protein expressed by the Pichia succeeded in expanding suspension cells of the normal plant type in cotton. There is no transcription difference between the mutant and the wild type on the GA synthesis genes like KO, KAO. Transcripts of the Expansins in the mutant was less than that in the wild-type at both 5th and 15th-leaf time point. Reversely, the mRNA of the GA receptor gene GID and DELLA were more in the dwarf, especially, the GID which was 60 times more at the 15th-leaf time point. Meanwhile the oxidase genes in the GA synthesis pathway were slightly high in the mutant, maybe it was caused by feedback regulation.3. The promoter region of the three Expansin genes was obtained by using genome walking technique. These genes were related to GA, and element of TAACAAA was found in the promoter region of these genes. A MYB gene interaction with this element was screened by using yeast one-hybrid screening approach. The in situ hybridization showed that this gene expressed in the same cell as that of expansion gene. The MYB gene sequence was homology with the GAMYB of other species. And the complementary experiments in vivo in tobacco show that the gene can be used as miR159 targets. Abundant studies have showed that the gibberellin regulated GAMYB and miR159 expression. The above shows that some expansin which directly role in the cell wall were regulated by gibberellins and GAMYB. The Expansin gene family was one of the gibberellin signal transduction terminal protein. Gibberellin synthesis was hampered in AS98, resulting in Expansin was reduced, and then, cell wall expansion was affected, causing dwarf finally. |
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