Functional analysis of zoysiagrass chitinase genes
- Abstract
- In this study, we isolated two full-length chitinase genes, class I chitinase (Zjchi1) and class II chitinase (Zjchi2) from zoysiagrass. Structural property and expression analysis of these genes was performed. Furthermore, the two chitinases were overexpressed in Escherichia coli and purified for assessment of their in vitro antifungal activity. Also, we generated transgenic zoysiagrass that overexpressed the class II chitinase gene (Zjchi2) via Agrobacterium-mediated transformation from zoysiagrass calluses, although its monocot and plant regeneration is normally difficult (Toyama et al. 2003; Poeaim et al. 2005). Southern blot hybridization and polymerase chain reaction (PCR) were performed to confirm the transgenes insertion in the transgenic zoysiagrass. Furthermore, the antifungal activity was investigated to examine the disease resistance of transgenic zoysiagrass to R. solani AG2-2 (IV). To our knowledge, this study is the first to verify the antifungal activity of a class II chitinase in transgenic zoysiagrass.
- Issued Date
- 2017
- Awarded Date
- 2017. 2
- Type
- Dissertation
- Alternative Author(s)
- Ji-Nam Kang
- Department
- 대학원 생명공학과
- Advisor
- 이효연
- Table Of Contents
- Table index 2
Figure index 3
1. Introduction 4
2. Material and methods 7
2.1. Plant materials and cloning of chitinase genes 7
2.2. Expression analysis of chitinase genes 8
2.3. Vector construction for recombinant (r) chitinases 8
2.4. Overexpression in E. coli and purification of r-chitinases 9
2.5. Antifungal activity of purified r-chitinases 10
2.6. Preparation of the binary expression vector 11
2.7. Development of transgenic zoysiagrass 12
2.8. PCR and Southern blot analysis of the transgenic zoysiagrass 13
2.9. Analysis of transgenic zoysiagrass gene expression 14
2.10. Phenotype analysis of transgenic zoysiagrass 14
2.11. In vitro, R. solani inoculation and detached leaf analysis 15
2.12. Soil-based infection analysis 15
3. Results 19
3.1. Sequence and structure of zoysiagrass chitinase genes 19
3.2. Expression analysis and antifungal activity of chitinases 23
3.3. Production and verification of transgenic zoysiagrass 29
3.4. Molecular analysis of the transgenic zoysiagrass 31
3.5. Morphology analysis of transgenic zoysiagrass 35
3.6. Antifungal activity analysis of transgenic zoysiagrass 38
4. Discussion 45
5. Conclusions 52
6. References 53
- Degree
- Doctor
- Publisher
- 제주대학교 일반대학원
- Citation
- 강지남. (2017). Functional analysis of zoysiagrass chitinase genes
- Appears in Collections:
- Faculty of Biotechnology > Molecular Biotechnology
- Files in This Item:
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