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Function Analysis Of Kinesin Isoform AtKP1 In Arabidopsis Thaliana

Posted on:2006-09-25Degree:DoctorType:Dissertation
Country:ChinaCandidate:X Y LiFull Text:PDF
GTID:1100360152980544Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
Kinesin is a kind of microtubule-dependent motor proteins that constitute a superfamily with many isoforms. Members of the kinesin superfamily perform many diverse cellular functions, such as transport of membrane organelles, mitosis, meiosis, microtubule dynamics and signal transduction etc. The study on kinesins in plants is focus on cell polar growth and cytokinesis. Functions of kinesin isoforms show diversity, so the study on special functions of kinesin isoforms in Arabidopsis would provide valuable information for understanding the function of the kinesin superfamily and the development mechanism of the Arabidopsis. AtKPl is a gene with characteristics of kinesin, which had been cloned from the stem of Arabidopsis in our laboratory. This study is focus on expression of kinesin isoform AtKPl and its biological function, and the results were as follows:Expression of AtKPl in different organs of Arabidopsis was analyzed by semi-quantitative RT-PCR, and the result demonstrated that AtKPl was expressed widely in young organs. In order to know the expression activity of AtKPl in vivo, the promoter region of AtKPl was cloned. Analysis by PLACE with Internet software indicated that the promoter region had not only basic elements like TATA box and CAAT box, but also auxin-response element. The promoter region was fused to gus (prokpl-gus) , and transformed into Arabidopsis. GUS expression pattern in transgenic Arabidopsis was analyzed. The results demonstrated that GUS was expressed mainly in young leaf trichomes and young stele, suggesting that AtKPl may be involved in development of stele and leaf trichome .Prokpl-gus transgenic plant were exposed to environment stress and auxin (10 u.M ABA, 1 mM IAA, 300 mM NaCl). Expression of AtKPl obviously responded to IAA, but not to ABA and high salt. Based on semi-quantitative RT-PCR analysis, AtKPl expression was responded only after induced for 48 h with IAA, indicating that AtKPl is a late response gene of IAA. To further reveal the function of AtKPl, mutant of AtKPl T-DNA insertion was confirmed by PCR, southern blotting, northern blotting, and its phenotype was observed. The germination of mutant seeds showed resistance to IAA, which suggested that AtKPl may be involved in IAA physiological process, but deletion of AtKPl didn't affect the change of the early response gene IAA2 JAA16 ; In normal growth conditions, the deletion of AtKPl didn't affect the normal growth and the development of Arabidopsis plants, suggesting that the function of AtKPl may be substituted by other homologous gene.KatD is another kinesin isoform with high identity to AtKPl. To know if KatD could substitute AtKPl, we cloned the promoter region of katD. With gus as reporter gene, expression pattern of KatD in Arabidopsis was analyzed. KatD had high expression in anther. Its strong expression was also occurred in young leaf trichome and young vasculature, similar to that of AtKPl.katD T-DNA insertion mutant was confirmed by the method of PCR and semi-quantitative RT-PCR. Observation of phenotype demonstrated that deletion of katD didn't affect normal development of Arabidopsis plant, suggesting that the function of KatD may be substituted with that of its homologous gene. KatD mutant was crossed with AtKPl mutant, and the double mutant was obtained. Primary observation to T2-generation showed that double mutant plant had obvious dwarf phenotype, but had no defect in leaf trichome and vasculature. The T3-generation double mutants were waiting to be observed in detail.
Keywords/Search Tags:AtKP1, gene expression, IAA, KatD, single mutant, double mutant
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