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Cloning And Preliminary Function Analysis Of A Gene Related With Pleiotypic Mutant Of Arabidopsis Thaliana

Posted on:2007-08-13Degree:MasterType:Thesis
Country:ChinaCandidate:P LiFull Text:PDF
GTID:2120360182494340Subject:Cell biology
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A pleiotypic mutant named 155 was isolated, utilizing promoter trap vector to transform Arabidopsis. Its alterations of phenotype and structure have been further studied at whole, organic and cellular levels. By means of backcross between wildtype and the mutant, its genetic background has been analyzed. Moreover, the trapped gene has been cloned and located through molecular techniques. Based on all these aspects, the expression pattern and functions of the trapped gene has been discussed.1. Noticeable pleiotropic phenotypes were associated with the mutant, including rounded cotyledons, shortened hypocotyls and total length, smaller rosette leaves with shortened petioles and downward leaf tip, shorter and thicker siliques, less internodes and delayed flowering time.2. Histological analyses in 155 plants demonstrate that 1) CZ of 10-day-old seedlings in 155 was almost flat, which was not as vaulted as C24. 2) At day 21, the CZ was vaulted, but the PZ (peripheral zone) lost its original uphill hogback. Besides, nonanticlinal cell divisions were observed in mutant tunica L2 layer. 3) Observations at day 30 reveal the transition from vegetative to reproductive growth was delayed (for about 7 days) in the mutant.3. Cotyledons of the mutant had a disordered vascular network with additional unclosed top loop and 2-6 free vein endings, which emanated from middle and lateral veins.4. Gus assay of SAM shows blue staining specifically expressed in rib zone and leaf primordium in the mutant.5. Genetic analysis indicates that the mutant phenotype in 155 was caused by a single, recessive, nuclear mutation.6. The physiological results reveal that 155 was less sensitive to exogenous auxin and gibberellin than C24, but the gross trend was similar in both 155 and C24.7. By using TAIL-PCR, the trapped gene has been cloned and the located on chromosome IV, and its property was related to HSP101.8. RT-PCR was performed in normal growth conditions, displaying the expression level of the trapped gene was identical in both C24 and 155. However, the expression level of the same gene wasn't detected apparently in 155 after heat shock at 37℃. Primary heat shock treatmentdemonstrates the mutant was noticeably sensitive to comparatively high temperature because the growth of the mutant was seriously inhibited which could not transit to reproductive phase from vegetative.In conclusion, the T-DNA insertion indeed caused the functional alterations of the trapped gene, and further affected a series of changes involving in morphology, structure and development, which reveals the protein related to HSPlOl encoded by the trapped gene may play an important role in the signal pathway which is necessary for regulating normal growth and development of Arabidopsis, through which SAM development and vascular patterning formation as well as the morphogenesis of plants are affected.
Keywords/Search Tags:Arabidopsis, T-DNA insertion, auxin, vascular patterning, SAM, heat shock protein, TAIL-PCR
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