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title page
Contents
요약 11
ABSTRACT 15
Introduction 19
Materials and methods 22
1. Rice transformation 22
2. Confirmation of transgene transmission and expression 22
3. Analysis of transgene integration site 23
4. Pi deficiency treatment and Pi uptake assay 25
5. Field performance test 26
6. Chemical analysis 26
7. Analysis of CO₂ assimilation and chlorophyll contents 27
8. Measurement of physicochemical property 27
9. Statistical analysis 28
10. Expression profile analysis 28
Results and discussion 30
1. Development of advanced generation of NtPT1 transgenic rice 30
2. Integration site of the transgene 30
3. Pi uptake and accumulation 31
4. Agronomic characteristics 35
5. Mineral nutrients contents and CO₂ assimilation 36
6. Grain quality 39
7. Response of genome expression profile to P-deficiency 40
General discussion 46
Figures 54
Tables 73
REFERENCES 93
Acknowledgements 99
Table 1. Cycling conditions used for TAIL-PCR. 73
Table 2. Yield-related characteristics of the T₃ transgenic lines and the control variety, Dongjinbyo. 74
Table 3. Contents of mineral elements and proteins in the T₃ transgenic lines and a control variety, Dongjinbyeo. 75
Table 4. Correlation between P and other mineral contents. 76
Table 5. Variations and varietal differences of pigmentation and palatability values in the T₃transgenic and control lines and correlations between P contents and other traits. 76
Table 6. Variations and varietal differences of physicochemical properties in the T₃transgenic and control lines and correlation between P contents and other traits. 77
Table 7. Variations and varietal differences of quality evaluation value, protein, amylose, fatty acids content in brown grains of the T3 transgenic and control lines and correlations between P contents and other traits. 77
Table 8. Variations and varietal differences of quality evaluation value, protein, amylose, fatty acids content in milled grains of the T₃transgenic and control lines and correlations between P contents and other traits. 78
Table 9. Variations and varietal differences in the texture of milled grains of the T₃transgenic and control lines and correlations between P contents and other traits. 78
Table 10. Functional classification of genes differentially responded to the P conditions by cluster of orthologous groups (COG) analysis. 79
Table 11. List of genes that are up-regulated or down-regulated over two-fold in the transcription level at high-P and low-P conditions both in the T₃transgenic and control plants. 80
Table 12. List of genes that are up-regulated or down-regulated over two-fold in the transcription level at high-P and low-P conditions only in the control plants. 86
Taele 13. List of genes that are up-regulated or down-regulated over two-fold in the transcription level at high-P and low-P conditions only in the T₃transgenic plants. 88
Table 14. Genes involved in P transport, metabolism and Pi signalling whose expression is up-regulated or down-regulated in the transcription level at high-P and low-P conditions in the control and T₃transgenic plants. 90
Table 15. Genes involved in carbohydrate metabolism whose expressioin is up-regulated or down-regulated in the transcription level at high-P and low-P conditions in the control and T₃transgenic plants. 91
Fig. 1. Costruction of NtPT1 expression cassette using the binary vector pGA 1611. pUbi, maize ubiquitin promoter: Hyg, Hygromycin phosphotansferase: Tnos, polyadenylation signal of the nopline synthase gene: CaMV35S, 35S promoter of... 54
Fig. 2. Specific primers (SP) used for TAIL-PCR. The primer sets for the pGA1611 vector are designated SP1, SP2, and SP3. In addition, SPR primer was designed to select specific clones, and Eco RV site is festriction enzyme site for select specific clones. 55
Fig. 3. Schematic diagram of TAIL-PCR contrasting the amplification of target with nontarget products. Boldface segments denote the specific primer (SP), and small opern rectangles denote the arbitrary degenerate primer (AD). 56
Fig. 4. Develoment of NtPT1 transgenic plants. Transgenic rice calli (A), shoot (B) and root (C) regeneration, T₁ transgenic plants gowing in pots filled with typical paddy soil in a glasshouse (D).... 57
Fig. 5. Southern (A) and Northern blot analysis (B) of NtPT1 transgene in the transgenic T₃ and non-transgenic rice plants. Lane 1, non-transgenic plant; Lane 2-28, transgenic lines 1-7-1, 1-7-2, 1-7-4, 1-7-6, 1-7-8, 1-7-12, 1-7-15, 1-9-1, 1-9-2,... 58
Fig. 6. Agarose gel analysis of secondary and tertiary TAIL-PCR products amplified by specific primer (SP2) and arbitrary degenerate (AD1) primer (A), SP2 and AD2 primer (B), SP2 and AD3 primer (C), SP3 and AD1 primer (D), SP3 and... 59
Fig. 7. Agarose gel analysis for the selection of products bordering the cloning vector arm. M; 1kb ladder (Promegea, USA), P; Positive control (pGA1611), N; Negative control (Dongjin byeo), 1; PCR products amplified by specific primer... 59
Fig. 8. Analysis of sequences of the specifically selected clone. Boldfont segments indicate the amplified target sequence, and italic boldfonts segments indicate the left T-DNA border. Region of SP3 primer shows the specific primer used for the tertiary PCR. 60
Fig. 9. Blast analysis of the sequence againt database in the National Center for Biotechnology Information (NCBI). 61
Fig. 10. The genomic region flankng the insertion site of T-DNA and NTPT1 sequence.... 62
Fig. 11. Analysis for binding domain elements and transcription factor on chromosome 3 by the Transcription Element Search System (TESS, http://www.cbil.upenn.edu/cgi-bin/tess/tess33?RQ=WELCOME). Pink rectangle... 63
Fig. 12. Southern blot analysis of NtPT1 transgene in the transgenic T₃ and non-transgenic rice plants. Lane 1, non-transgenic plant; Lane 2-5, transgenic lines 1-7-5, 1-7-6, 1-7-8, and 10-2-7, respectiviely. Genomic DNAs were= completely digested... 64
Fig. 13. Pi uptake rates of the plants grown at high-P and low-P conditions in a hydroponic culture system. 65
Fig. 14. P contents in the plants grown at high-P and low-P conditions in a hydroponic culture system. Four-week-old transgenic(1-7-4, 1-7-6, 1-7-8 and 10-2-7) and non-transgenic (CTR) plants were grown for a two week-period in... 66
Fig. 15. Performance of the agronomic traits of the nineteen transgenic lines and a control variety in a typical paddy field. Tramsgemoc (1-7-1, 4, 6, 8, 15, 1-9-1, 3, 10-2-7, 8, 10-8-1, 7, 59-5-4, 59-6-6, 59-6-6, 96-11-5, 96-12-4,122-14-5 and 122-18-3) and... 67
Fig. 16. CO₂ assimilation and chlorophyll contents of the nineteen transgenic lines and a control variety at 10 days before heading, heading, and matuaring period in a typical paddy field.... 68
Fig. 17. Content of mineral elements in the seeds of plants grown in a typical paddy field. Transgenic (1-7-8 and 122-14-5) and non-transgenic (CTR) plants were grown in paddy field containing 140 mg Pi/Kg. The columns indicated by the... 69
Fig. 18. Growth of transgenic and control plants at high-P and low-P conditions in a hydroponic culture system. Four-week-old transgenic line 1-7-8 (PT) and non-transgenic (CTR) plants were grown for 5 days in solutions containing 320 (high-p)... 70
Fig. 19. Categorical distribution of 3,691 genes showing differential expression under the low-P condition in the transgenic or control plants. Four-week-old transgenic (1-7-8) and non-transgenic plants were grown for 6 days in solutions... 71
Fig. 20. Hierarchical clustering of genes showing differential expression in response to low-P condition. Group A, genes down-regulated under low-p condition in both genotypes (1,525 genes); Group B, genes down-regulated or up-regulated... 72
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