VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------------- GRADUATION THESIS TITLE: CONSTRUCTION OF OVEREXPRESS VECTOR, PROMOTER VECTOR AND CRISPR/CAS9 VECTOR FOR OsGDPD13 GENE EDITING IN RICE HANOI – 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------------- GRADUATION THESIS TITLE: CONSTRUCTION OF OVEREXPRESS VECTOR, PROMOTER VECTOR AND CRISPR/CAS9 VECTOR FOR OsGDPD13 GENE EDITING IN RICE Student : KIEU TRINH VAN AN Student code : 637402 Faculty : BIOTECHNOLOGY Supervisor : TO THI MAI HUONG, PhD (USTH) NGUYEN THI THUY HANH, PhD (VNUA) HANOI - 2022 COMMITMENT I hereby declare that this is my research project under the guidance of To Thi Mai Huong, PhD and Nguyen Thi Thuy Hanh, PhD. The results and data in this thesis are truthful have not been published by anyone in any way. I confirm that all information and data from articles and sources of other authors contain full citations and references from official sources. I take full responsibility for this guarantee.
Hanoi, November 24th, 2022 Student Kieu Trinh Van An i ACKNOWLEDGEMENTS First and foremost, I would like to express my deep thanks to To Thi Mai Huong, PhD and Nguyen Thi Thuy Hanh, PhD, who helped and gave me many advices for my graduation thesis. I appreciate the dedicated instruction of To Thi Mai Huong, PhD, she and always made it easy for me to conduct the experiments I needed to finish this graduation thesis. I would like to express my sincere thanks to the Vietnam National University of Agriculture, the Faculty Board and lecturers in the Faculty of Biotechnology for creating an interesting learning environment and supplying me with priceless knowledge and experiences over the past four years. During the course of the thesis at the Plant Biotechnology Laboratory, University of Science and Technology of Hanoi (USTH), To Thi Mai Huong, PhD and lecturers of Life Science Faculty helped me complete my thesis and draw me a lot of experiences.
They are really dedicated to the profession, devote their best and have inspired me great inspiration in scientific research. I want to thank the administration of the University of Science and Technology of Hanoi for making it possible for me to work here once more and everyone who helped me finish my thesis. Finally, I would like to thank my family for always being supportive of me while I wrote my thesis and for always listening to me and offering me wise counsel. Hanoi, November 24th, 2022 Student Kieu Trinh Van An ii TABLE OF CONTENTS COMMITMENT .ii TABLE OF CONTENTS.
iii LIST OF TABLES. v LIST OF FIGURES .vi LIST OF ABBREVIATION. The role of phosphorus fertilizers for rice and its impact on the environment. The role of phosphorus fertilizers for rice.
Impact of phosphorus fertilizers on the environment. Genes related to the tolerance of rice to low-Pi condition. Family of GDPDs. Gateway cloning technique.
MATERIALS AND METHODS. Time and place of study. Genomic DNA, sgRNA. Vectors and primers.
Chemicals and reagents. OsGDPD13 Overexpress Cloning. OsGDPD13 Promoter Cloning. OsGDPD13 Cloning using CRISPR/Cas9 technology.
RESULTS AND DISCUSSION. OsGDPD13 overexpress cloning. coli OsGDPD13 plasmid. BP reaction and transformation in DH5α.
Check Gateway BP cloning by digestion and PCR method. OsGDPD13 promoter cloning. Amplify the promoter of the OsGDPD13 gene from gDNA of Kitaake. Ligation reaction and transformation in DH5α.
Check OsGDPD13 promoter cloning by digestion. Sequencing result of OsGDPD13 promoter region. OsGDPD13 Cloning using CRISPR/Cas9 technology. Check Gateway LR cloning by colony PCR.
Sequencing result of OsGDPD13 Cloning using CRISPR/Cas9 technology. 33 CHAPTER V: CONCLUSION AND RECOMMENDATION. 39 iv LIST OF TABLES Table 3. List of two gRNAs.
List of primers. Component of BP Clonase reaction. Composition of digestion for checking Gateway BP cloning. Composition of PCR reaction for checking Gateway BP cloning.
Composition for gradient PCR. PCR program for gradient PCR. Composition of blunt-end cloning. Composition of digestion for checking blunt-end cloning.
Composition of blunt-end cloning for pJET1.2/blunt- OsGDPD13 vector construction. Composition of LR Clonase reaction. Composition of colony PCR for checking Gateway LR cloning. 23 v LIST OF FIGURES Figure 2.
A schematic pathway of phospholipid degradation mediated by GDPD. Gateway cloning BP reaction for a single fragment. Gateway cloning LR reaction. Mechanism of CRISPR/Cas9 genome editing.
Schematic diagram of pCAMBIA5300 Overexpress Vector. Schematic diagram of pJET 1. Schematic diagram of pOs-Cas9 vector. PCR program for checking the Gateway BP cloning.
PCR program for checking the Gateway LR cloning. The digestion result of E. coli OsGDPD13 plasmid on agarose gel 1%. The colonies of E.
coli pC5300-OsGDPD13OE grown on LB agar + Kana50. The result of checking Gateway BP cloning by digestion on agarose gel 1%. The result of checking Gateway BP cloning by PCR on agarose gel 1%. The result of amplification promoter of the OsGDPD13 gene from gDNA of Kitaake on agarose gel 1%.
The colonies of E.2/blunt-OsGDPD13prom grown on LB agar + Amp100. The result of checking OsGDPD13 promoter cloning by digestion on agarose gel 1% .2/blunt-OsGDPD13 promoter vector .2/blunt-sgRNA vector. The result of checking OsGDPD13 Cloning using CRISPR/Cas9 technology by Colony PCR on agarose gel 1%. 32 vi LIST OF ABBREVIATION Abbreviations Definitions bp basepair Cas CRISPR-associated cDNA Complementary DNA CDS Coding sequence CRISPR Clustered Regularly Interspaced Short Palindromic Repeats crRNA CRISPR RNA DAG Diacylglycerol DNA Deoxyribonucleic Acid G-3-P sn-glycerol-3-phosphate gDNA Genomic DNA GDPD Glycerophosphodiester phosphodiesterase GPD glycerophosphodiester GWAS Genome-wide association study kb Kilobase mM Milimolar ng Nanogram NPC Non-specific PLC Ubi Ubiquitin P Phosphorus PA Phosphatidic acid PC Phosphatidylcholine PCR Polymerase Chain Reaction Pi Phosphate PI-PLC Phosphoinositides-specific phospholipase C PLC Phospholipase C PLD Phospholipase D PUE Phosphorus use efficiency vii RNA Ribonucleic Acid rpm Revolutions per minute sgRNA Single-guide RNA SNP Single nucleotide polymorphisms µg Microgram µl Microliter viii ABSTRACT Phosphorus (P) is an essential plant macronutrient, the low availability of P in most soils requires an additional phosphate fertilizer to the plants.
Depletion of non- renewable rock phosphate reserves and increases of phosphorus fertilizer price imposes serious limitation on crop production; therefore, it is important to develop P- efficient rice varieties. Glycerophosphodiester phosphodiesterases (GDPDs) have been suggested to play important roles in phosphate homeostasis. 13 OsGDPDs genes were identified in rice, and characterized their roles in P deficiency. However, the expression profile of OsGDPD13 gene was not described.
Therefore, to serve the functional study of OsGDPD13 gene, it is necessary to construct the overexpress vector, promoter vector and CRISPR/Cas9 vector for OsGDPD13 gene editing in rice. In this study, pC5300-OsGDPD13OE vector, pJET1.2/blunt-OsGDPD13 promoter vector and pOs-Cas9-sgRNA vector were constructed. In addition, created three strains of E. coli carrying overexpress vector, promoter vector and CRISPR/Cas9 vector of OsGDPD13 gene.
The result provides a foundation for development of transgenic rice plants from Kitaake rice variety which will be applicable to study P deficiency-dependent response and to improve rice's adaptability to P deficiency stress. ix CHAPTER I: INTRODUCTION 1. Introduction After maize and sugarcane, rice is the agricultural product with the third-highest global production. For almost half of the world's population, rice agriculture provides both food and economic security; however, due to both population growth and the effects of climate change, the current state of food security is turning into an urgent global issue.
Phosphorus (P) is a crucial macro-element for all living cells and is therefore crucial to agricultural production systems. Low levels of available P in many soils limit the potential yield and general biomass production. Through the use of P fertilizer, modern agricultural practice has attempted to solve this issue, and this strategy has significantly improved crop yields. However, because of the limited availability of P resources and its negative impacts on environment, it will be necessary to create new phosphate (Pi)-efficient rice varieties.
By using a thoroughly genotyped Vietnamese rice pane to evaluate the Pi deficiency response in rice, To et al. (2020) and Mai et al. This gene was also identified by Mehra and Giri (2016). The expression profile of the OsGDPD13 gene, however, was not available in Mehra's study.
In light of this, research project “Construction of Overexpress vector, promoter vector and CRISPR/Cas9 vector for OsGDPD13 gene editing in rice” was carried out. In this study, the OsGDPD13 gene and OsGDPD13 promoter were desired fragments that were inserted into donor vectors using the gene cloning technique. The ligation products were transformed into E. The pOs-Cas9-sgRNA vector was constructed and transformed into E.
From that design, it is applied for loss function, gain function and expression study in Vietnamese rice to better understand the function of the OsGDPD13 gene. Objective Successfully clone the OsGDPD13 gene and OsGDPD13 promoter to construct pC5300-OsGDPD13OE vector, pJET1.2-OsGDPD13 promoter vector and pOS- Cas9-sgRNA vector. Requirements ‐ The OsGDPD13 promoter amplification. ‐ Transformation of competent cell E.
coli strains with pC5300-OsGDPD13OE vector, pJET1.2-OsGDPD13 promoter vector and pOs-Cas9-sgRNA vector. ‐ Selection of colonies containing recombinant plasmids. 2 CHAPTER II: LITERATURE OVERVIEW 2. Role of phosphorus fertilizers for rice and its impact on the environment 2.
Role of phosphorus fertilizers for rice According to FAO (2022), rice is one of the four individual crops accounted for half the global production of primary crops in 2020 (0. Vietnam's total rice area in 2021 was 7.24 million hectares, with a yield of 60.6 quintals per hectare. Vietnam's rice industry has had a strong transformation towards implementing advanced rice production processes. Currently, most of the rice growing households that have applied the program “Mot phai, nam giam” (1 Must, 5 Reductions) which includes: must use certified rice varieties and reduce the amount of seed sowing, reduce fertilizer, reduce irrigation water, reduce the number of times to use pesticides, reduce post-harvest losses (Jackson et al.
In agricultural production, crop yield and production efficiency are the top goals. They depend on input factors such as plant varieties, cultivation processes, pest management,. in which, fertilizer is one of the important materials and is used in a fairly large amount every year. Especially for rice, fertilizer plays a particularly important role in intensive farming to increase rice yield.
One of the elements required for plant metabolism is phosphorus (P), which is found in many of the essential organic compounds for plants. P participated in many significant organic compounds and the majority of the metabolism of plants after entering plants as inorganic compounds via primary assimilation by the root system. P encourages the use and synthesis of nitrogen in plants, stimulates the growth of roots, improves the quality and yield of rice. Impact of phosphorus fertilizers on the environment P in manure and waste was once returned to the soil as part of a natural cycle to help with crop production.
Industrial agriculture moves food across the globe for processing and consumption, disrupting the natural cycle that returned phosphorus to the soil through the decomposition of plants. To increase the nutrients in the soil, fertilizer must often be applied continuously. In sandy-textured soils, this practice 3 will eventually lead to an increase in soil P accumulation, the danger of off-site movement, and leaching (Mylavarapu, 2017). P’s major source in current use, phosphate rock, is a non-renewable resource.
The projections by FAO in 2009 show that feeding a world population of 9.1 billion people in 2050 would require raising overall food production by some 70 percent between 2005/07 and 2050. Production in the developing countries would need to almost double (FAO, 2009). P is crucial to global food security. Yet, there are no international organizations or regulations that manage global P resources.
Moreover, climate change will affect the demand for P because agriculture will bear the brunt of changing weather patterns. Most P is lost or wasted.