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The role of Cleavage and Polyadenylation Specificity Factor (CPSF) gene in Brassinosteroid signal transduction in Arabidopsis thaliana

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Author(s)
딜리프러사드퍼우디얄
Issued Date
2008
Keyword
activation tagging|Arabidopsis|BRs signaling|Brassinazole
Abstract
Brassinosteroids (BRs), a class of plant steroid hormones, are ubiquitously involved in regulating normal growth processes. The hormone controls flowering, leaves shape, color, male fertility, cell elongation and vascular differentiation. Genetic assessments explored a number of genes showing their role in BRs response pathways. We have screened the activation tagged mutants that have severed growth defects and respond to a BRs biosynthetic inhibitor, brassinazole (Brz). Brassinazole is an inhibitor of active BRs that develops characteristic light grown phenotype under dark. The mutant shows taller, dark green, late flowering, short petioles, short and/or curved siliques had fewer seeds with low germination. The mutant had hard texture in maturity. Fruiting was appeared successionally in the mutant having various stages of maturity. TAIL-PCR, genotyping PCR and segregation ratio of T2 plants revealed a single T-DNA insertion at the first exon of Cleavage Polyadenylation Specific Factor (CPSF) gene in chromosome III that encodes a subunit C-terminal domain-containing protein; similar to DDB1A (UV-damaged DNA-binding protein 1A) in Arabidopsis thaliana. To see response of the gene with other BR related mutants, we crossed the mutant between det2, a BRs biosynthetic mutant, bin2 and bes1D. We also observed the sense and antisense expression effect of the gene in phenotype. Real Time RT-PCR results showed that there was no alteration in expression level of neighboring genes of the T-DNA insertion. Hypocotyls length of Brz-treated mutant seedlings under dark measured nearly two fold longer than wild type Columbia 0 revealing involvement of cpsf in BRs signal transduction. The crossing results reflected that the T-DNA insertion mutation of CPSF related to BR perception and it regulate normal growth process through involving in BRs signaling pathways in Arabidopsis. Also cpsf knockdown or overexpression creates defective siliques and might develop lethality in Arabidopsis.
Alternative Title
애기장대에서 브라시노스테로이드 신호전달에 관여하는 CPSF 유전자의 작용기작
Alternative Author(s)
Dilli Prasad Paudyal
Affiliation
조선대학교 대학원
Department
일반대학원 유전자과학과
Advisor
정현숙
Awarded Date
2008-08
Table Of Contents
1 INTRODUCTION = 1
1.1 GROWTH PROCESS AND ITS CONTROL = 1
1.2 THE HISTORY OF PLANT HORMONES = 2
1.3 THE HISTORY OF BRASSINOSTEROIDS = 2
1.4 NATURAL OCCURRENCE OF BRASSINOSTEROIDS IN PLANTS = 6
1.5 BIOSYNTHESIS = 7
1.6 PHYSIOLOGICAL ACTION OF BRASSINOSTEROIDS = 9
1.7 MOLECULAR GENETICS OF BRASSINOSTEROID ACTION = 12
1.7.1 Brassinosteroid signal transduction = 12
1.7.2 Brassinosteroid regulated gene expression = 14
1.8 CPSF GENE AND ITS ROLE = 16
1.9 AIM OF THE WORK AND EXPERIMENTAL DESIGN = 17
2 MATERIALS AND METHODS = 19
2.1 GROWTH MEDIA = 19
2.1.1 Media for plant culture = 19
2.1.2 Bacteria culture medium = 20
2.2 SEED STERILIZATION = 20
2.3 SOWING SEEDS = 21
2.4 GROWTH CONDITION = 21
2.5 CELL PREPARATION AND TRANSFORMATION = 21
2.5.1 E. coli transformation = 21
2.5.2 Heat-shock transformation of competent E. coli cells = 22
2.5.3 Electroporation = 22
2.5.4 Transformation of Agrobacterium tumefaciens = 22
2.5.5 Cold shock transformation of competent Agrobacterium tumefaciens cells = 23
2.6 SCREENING ACTIVATION-TAGGED COL-0 AND GENOTYPING = 23
2.7 DOUBLE MUTANT GENOTYPING = 24
2.8 TOTAL RNA ISOLATION AND CDNA SYNTHESIS = 24
2.9 FULL LENGTH CDNA CONSTRUCTION = 25
2.10 AGROBACTERIUM-MEDIATED TRANSFORMATION INTO PLANTS.= 27
2.11 SOME PRIMERS USED = 27
3 RESULTS = 29
3.1 ACTIVATION TAGGED MUTANT SCREENING = 29
3.2 PLANT MORPHOLOGY = 31
3.3 T-DNA INSERTION LINE SELECTION AND ITS ORIENTATION = 33
3.4 GENE EXPRESSION ANALYSIS = 36
3.5 HORMONE DOSE RESPONSE AND HORMONAL CROSSTALK = 38
3.5.1 Hormone dose response = 38
3.5.2 Hormone cross talk = 42
3.6 CROSSING WITH BRASSINOSTEROID RELATED MUTANTS = 42
3.6.1 Morphology of crossed lines = 46
3.7 GERMINATION IMPAIRMENTS IN MUTANT AND ITS CROSSED LINES = 49
3.8 CPSF SENSE AND ANTISENSE EXPRESSION CONSTRUCT = 51
4 DISCUSSIONS = 62
5 REFERENCE = 71
ACKNOWLEDGEMENT = 85
Degree
Doctor
Publisher
조선대학교 대학원
Citation
딜리프러사드퍼우디얄. (2008). The role of Cleavage and Polyadenylation Specificity Factor (CPSF) gene in Brassinosteroid signal transduction in Arabidopsis thaliana.
Type
Dissertation
URI
https://oak.chosun.ac.kr/handle/2020.oak/7271
http://chosun.dcollection.net/common/orgView/200000236495
Appears in Collections:
General Graduate School > 4. Theses(Ph.D)
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