4 research outputs found
Transgenic lines of melon (Cucumis melo L. var. makuwa cv. “Silver Light”) expressing antifungal protein and chitinase genes exhibit enhanced resistance to fungal pathogens
東方甜瓜 (Cucumis melo L. var. makuwa cv. “Silver Light”)為一重要之水果作物,其主要分佈於熱帶及亞熱帶地區。然而,真菌引起的疾病會造成其產量下降。
本研究將抗真菌蛋白(AFP)與幾丁質酶(CHI)之融合基因轉殖入東方甜瓜中,探討該基因轉殖作物對於真菌枯絲核菌(Rhizoctonia solani)及尖孢鐮刀菌(Fusarium oxysporum)所引起疾病之抗性。轉殖方式乃利用農桿菌LBA4404(Agrobacterium tumefaciens strain LBA4404)行之,該農桿菌含有抗真菌蛋白(AFP)與幾丁質酶(CHI)基因,其啟動子為椰菜嵌紋病毒35S啟動子(Cauliflower mosaic virus, CaMV),篩選標記為新黴素磷酸轉移酶(neomycin phosphotransferase, nptII)基因。
利用農桿菌將pBI121- AFP- CHI基因轉殖入懸浮培養中之東方甜瓜子葉體後,再建立植物培養系統。以聚合酶鏈反應(polymerase chain reaction, PCR)確認基因是否轉殖入。南方墨點法(Southern blot)之結果確認PCR陽性轉殖株之AFP-CHI融合基因確實被轉殖入。RT-PCR分析結果則確認於個別基因轉殖株之AFP-CHI融合基因確實有表現。 西方墨點法(Western blot)分析結果顯示CHI蛋白累積於葉片上。分離分析(segregation analysis)顯示該基因可被遺傳至第一子代(T1)。
研究結果指出,本系統可有效建立具抗枯絲核菌和尖孢鐮刀菌之AFP-CHI基因轉殖甜瓜。The oriental melon (Cucumis melo L. var. makuwa cv. “Silver Light”) is an important fruit crop in the tropical and subtropical regions. However, oriental melon production is severely decreased by fungal diseases.
In this study, antifungal protein (AFP) and chitinase (CHI) fusion genes were introduced into oriental melons to control fungal diseases caused by Rhizoctonia solani and Fusarium oxysporum. Transformation of oriental melon (Cucumis melo L. var. makuwa cv. “Silver Light”) with Agrobacterium tumefaciens strain LBA4404 containing antifungal protein (AFP) and chitinase (CHI) fusion genes under the control of the cauliflower mosaic virus (CaMV) 35S promoter and neomycin phosphotransferase (nptII) gene as a selectable marker was performed. Cotyledon explants of oriental melon were inoculated by Agrobacterium suspensions with pBI121–AFP–CHI and cultured in a regeneration medium. After regeneration, genomic DNA polymerase chain reaction (PCR) was conducted to confirm the presence of putative transgenic shoots. Southern blot analysis confirmed that the AFP–CHI fusion gene was incorporated into the genomic DNA of the PCR–positive lines. RT–PCR analysis showed that the AFP–CHI fusion gene was expressed in the individual transgenic lines. Western blot analysis revealed the accumulation of CHI protein in leaves. A segregation analysis of the T1 generation confirmed the inheritance of the transgene. Our results demonstrated that the AFP–CHI fusion gene was effective in protecting the transgenic melon plants against fungal disease caused by Rhizoctonia solani and Fusarium oxysporum.TABLE OF CONTENTS Page
Acknowledgments I
Absract (Chinese version) II
Absract III
Table of contents IV
LIST OF FIGURES VII
LISTOF TABLES VIII
Chapter 1: Literatures review: 1
1.1 Botany of Cucumis melo 1
1.2 Biotechnology and in genetic engineering in plant 3
1.3 Tissue culture of Cucumis melo 4
1.4 An Overview of Agrobacterium-mediated transformation 6
1.4.1 Biology of Agrobacterium-tumefaciens 6
1.4.2 Tumor inducung (Ti) plasmid 8
1.4.3 Transfer DNA (T-DNA) of Ti plasmid 9
1.4.4 Virulence genes 10
1.4.5 Plant transformation via Agrobacterium tumefaciens 11
1.4.6 Binary vectors of Ti plasmid 12
1.5. Agrobacterium-mediated transformation of Cucumis melo 15
1.6 Selectable marker genes 16
1.7 Antifungal proteins 17
1.8 Chitinases and their use in genetic engineering 18
1.8.1 Chitinases 18
1.8.2.Chitinases in nature 19
1.8.3 Plant chitinases and their classification 20
1.8.4 Role of chitinases in plant defense against pathogens 25
1.8. 5 Expression of AFPCHI genes in transgenic plants 27
Chapter 2. Objectives 29
Referencess 30
Chapter 3. Enhanced tolerance of transgenic melon expressing chitinase and antifungal protein to fungal pathogen
3.1.Introduction 49
3.2. Material and Methods 51
3.21 Plant material 51
3.2.2 Tissue culture media and culture conditions 51
3.2.3 Agrobacterium strain and vector construction 52
3.2.4 Infection of explants and Co-cultivation 53
3.2.5 Selection of explants 53
3.2.6 Elongation of explants 54
3.3 Molecular analysis 55
3.3.1 Genomic DNA isolation 55
3.3.2 DNA quality measurement 56
3.3.3 Polymerase Chain Reaction (PCR) 57
3.3.4 Southern blot analysis 58
3.3.5 Total RNA isolation 59
3.3.6 Measuring RNA concentration 60
3.3.7. RT-PCR 61
3.3.8 Formaldehyde Agarose Gel Electrophoresis 62
3.3.9 Western blot analysis 63
3.4 Disease resistance assay with R. solani and F. oxysporum 64
3.5 Segregation analysis 65
4. Result 66
4.1 Production of transgenic melon lines 66
4.1.1 Callus induction 66
4.1.2 Shoot development 66
4.1.3 Acclimization 67
4.2 Molecular analysis 68
4.2.1 Integration of transgene host genome 68
4.2.2 Expression of transgene 68
4.3 Antifungal assay 69
4.3.1 Disease resistance assay with R. solani 69
4.3.2 Disease resistance assay with F. oxysporum 69
4.4 Segregation analysis 70
5. Discussion 71
Chapter 4 Conclusion 75
Referencess 98
LIST OF FIGURES
Chapter 1
Figure 1 Agrobacterium-mediated plant transformation process 11
Figure 2 The classification of plant chitinase 24
Chapter 3
Figure 3.1 Partial map of the plasmid vector used for transformation of Cucumis melo L. var. makuwa cv. “Silver Light” AFP-CHI gene isolated from papaya.. 76
Figure 3.2 Inoculation procedure of Rhizoctonia solani 77
Figure 3.3 Inoculation of procedure of Fusarium oxysporum 78
Figure 4.1 Infection procedure of Agrobacterium tumefaciens 79
Figure 4.2 Shoot and root development of transgenic melon lines 80
Figure 4.3 Acclimization of transgenic melon lines 81
Figure 4.4 PCR analysis of transgenic melon lines 82
Figure 4.5 Southern blot analysis of individual transgenic lines 83
Figure 4.6 Fungal resistance test of transgenic melon expressing AFP-CHI gene against R. solani 84
Figure 4.7 Plant survival of the wild-type, P1 and individual transgenic lines inoculated with R. solani, measured 7 days after inoculation 85
Figure 4.8 Representative photographs of the wild type, P1, B4, D1, and M1 transgenic plants grown in greenhouse at 15 days after inoculation F. oxysporum 86
Figure 4.9 RT-PCR analysis of individual transgenic lines 87
Figure 4.10 Western blot analysis individual transgenic lines challenged with R. solani 88
LIST OF TABLES
Chapter 1
Table 1. Plant chitinases that have been isolated and cloned 21
Chapter 3
Table 3.1 The compositon of tissue culture medium 89
Table 3.2 Buffer and Solutions of Genomic DNAisolation 90
Table 3.3 Buffer and Solutions of Southern blot analysis 91
Table 3.4 Wash solutions of Southern blot analysis 92
Table 3.5 Compositon of formaldyehde agarose gel electrophoresis 93
Table 3.6 1X FA Gel running buffers 94
Table 3.7 5X FA Gel running buffers 95
Table 4.1 Resistance of individual transgenis lines after inoculation with F. oxysporum 96
Table 4.2 Segregation analysis of individual transgenic lines selection on MS medium with supplemented on kanamycin 9
Temperature-Dependent Demography of <I>Aulacaspis yasumatsui</I> (Hemiptera: Diaspididae)
2nd International Conference on Agricultural and Biological Sciences (ABS 2016), Shanghai, China, July 23-26, 2016 Abstracts
Table of contents
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14 Research on the seed-like Fruits of Subg. Sclarea of Salvia of Labiatae in China
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