VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------------***-------------- GRADUATION THESIS TITLE: CHARACTERIZATION OF MUTATIONS IN THE OsDSG1 PROMOTER OF RICE LINES INDUCED BY THE CRISPR/CAS9 SYSTEM Hanoi - 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -------------***-------------- GRADUATION THESIS TITLE: CHARACTERIZATION OF MUTATIONS IN THE OsDSG1 PROMOTER OF RICE LINES INDUCED BY THE CRISPR/CAS9 SYSTEM Student’s name : TRAN HAI PHUONG Class : K63CNSHE Student’s code : 637262 Supervisor : Dr. DO TIEN PHAT Dr. BUI THI THU HUONG Major : BIOTECHNOLOGY Hanoi - 2022 COMMITMENT I hereby declare that the entire results of the thesis were done by myself. Data and results published in essay were completely truthful, accurate and have not been published in any other works or research.
Hanoi, 1th December, 2022 Student Tran Hai Phuong i ACKNOWLEDGEMENTS First and foremost, I would like to express my deep gratitude to the lecturers in VNUA who have supported me in theory and practice throughout the past 4.5 years of study and especially, Dr. Bui Thi Huong helped me through this thesis. Words cannot express how grateful I am for her unconditional support and belief during the toughest period of my life. Huong’s help, this thesis would not be able to be completed.
In addition to this, I would like to extend my thanks and appreciation to the executive board of the (IBT), especially Dr. Do Tien Phat, for their critical advice and constructive feedback on my experiments and thesis writing. Being a graduate student, much of help and support from Mr. Doai, who wholeheartedly guided me with professional knowledge and practical skills, expanding and improving my knowledge.
I am very happy to have the chances to meet and work with the staff in Plant Cell Biotechnology. I could not fill my work and complete my duties without their help. In addition, the enthusiasm and thoughtfulness of the staff at the plant cell technology department were also a great spiritual gift to me. Last but not least, I would like to express my thanks to my family for their unconditional sacrifices and friends who always stay with me and encourage and inspirate me through the process of learning and studying.
With warm regards, Hanoi, December, 2022 Student Tran Hai Phuong ii LIST OF CONTENTS COMMITMENT. ii LIST OF CONTENTS. iii LIST OF TABLES. v LIST OF FIGURES .vi LIST OF ABBREVIATIONS.
Aim of the study. General introduction of rice (Oryza sativa L. Rice (Oryza sativa L. Rice production in Viet Nam.
Abiotic stress and abiotic stress resistance in rice. Overview of the Ring finger E3 ligases (OsDSG1) gene in rice. Gene OsDSG1, a Ring finger E3 ligases, to create stress-resistant transgenic plants. The role of DSG1 on growth and development in rice.
The role of DSG1 in salinity and drought tolerance in rice. Application of CRISPR/Cas9 in generating mutations on the DSG1 promoter. MATERIALS AND METHODS. Materials and chemicals.
Check the presence of editing system in transgenic T0 and T1 plants. Total DNA extraction. Detection of transgenes in T0 and T1 plants based on Hygromycin. Analysis of rice lines T0 carrying mutations of the target gene.
Mutation detection by analysis of DNA Heteroduplexes. Amplify DSG1 promoter region. Cloning of DSG1 promoter. Sequencing and analysis of sequence results.
Inheritance of OsDSG1 promoter mutations at the T1 generation. RESULTS AND DISCUSSION. Analysis of DSG1 promoter mutation in T0 plants. Detect transgenes in T0 plants.
Mutation detection of DSG1 promoter in T0 plants. Amplify DSG1 promoter region. Cloning of DSG1 promoter. Sequencing and analysis of sequence results in T0 generation.
Inheritance of OsDSG1 promoter mutations at the T1 generation. Analysis of mutations in T1 plants. Sequencing and analysis of sequence results in T1 generation. Detect transgenes in T1 plants.
Mutations induced in the DSG1 promoter in the T0 plants. Inheritance of DSG1 promoter mutations at the T1 generation. 43 iv LIST OF TABLES Order Name of table Page Table 3. Components of the PCR reaction.
Primers were used in PCR. Primers were used in PCR. Components of the PCR reaction. Investigate the inheritance of mutant alleles in the T1 generation.
37 v LIST OF FIGURES Order Name of figure Page Figure 2. Working model of ABA-signaling pathway in germinating rice seeds. Phenotypes of OsDSG1. Abiotic stress responses.
Model of promoter showing core promoter, proximal promoter, and distal promoter regions. (Carlos M et al. Schematic representation of CRISPR/Cas9-mediated targeted mutagenesis in the rice OsDSG1 gene. Electrophoresis of PCR product of with Hpt F/R primers.
Detection of mutations in OsDSG1 promoter in T0 plants using heteroduplex analysis. Electrophoresis of PCR products amplified from OsDSG1 promoter in T0 plants, using PrDSG1-F1667/R260. Electrophoresis of PCR products to determine the presence of OsDSG1 promoter in T0 plants after being transformed. Sequence alignment of DSG1 promoter alleles in T0 plants.
PCR detects the mutation DSG1 promoter of the T1 progeny were derived from three T0 plants: 2. Cas9/gRNA-induced homozygous mutations in the DSG1 promoter found in T0 and T1 mutant population. Electrophoresis of PCR product to detect transgenes in T1 plants. 39 vi LIST OF ABBREVIATIONS Abbreviation Definition bp Base pair CH3COONa Sodium acetate DNA Deoxyribonucleic acid E.
coli Escherichia coli EDTA Ethylenediaminetetraacetic acid EtOH Ethanol F/R Forward and Reverse HCl Hydrochloric acid IPTG Isopropyl β-D-1-thiogalactopyranoside kb Kilobase LB culture Lysogeny broth M Generuler Bertani (DNA marker) Mt Milion tons NaOH Sodium hydroxide OsDSG1 Oryza sativa Delayed Seed Germination 1 PCR Polymerase Chain Reaction ul Micro litte RNAi RNA interference SDS Sodium Dodecyl Sulfate t/ha Ton/ha WT Wild-type X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactoside vii ABSTRACT Drought and salinity stresses seriously affect rice plant growth and yield. The cultivation of salinity and drought tolerant cultivars was the most cost- effective and environmentally friendly approach for salinity control. In recent years, CRISPR/Cas9 systems have been widely used for target-site genome editing; however, their application for the improvement of eliterice cultivars has rarely been reported. Abscisic acid (ABA) was a plant hormone that regulates numerous aspects of plant growth, development, and stress responses.
In 2010, Park et al, demonstrated OsDSG1 gene from rice was a major regulator of ABA signaling in germinating seed that controls seed germination and stress responses. The RNA interference silencing of OsDSG1 plants were drought and salinity tolerance, due to the transcript levels of ABA signaling and responsive genes were significantly increased. However, mutant plants were delayed-germination and had shorter phenotype wildtype. Therefore, the application of the CRISPR-Cas 9 system to induce mutations on the DSG1 promoter will be a potential way to creat transgenic plants that are tolerant to environmental stress.
In this study, we identified and characterized mutations in the OsDSG1 promoter of rice lines induced by the CRISPR-Cas 9 system. Four of eleven T0 transgenic plants showed mutations in the targeted gene. These mutations of the DSG1 promoter, were passed to the T1 generations. In addition, we found 2 of 16 tested T1 plants carrying homozygous mutations and free of transgenes.
This result indicates that the CRISPR/Cas9- mediated mutations in the DSG1 promoter were successfully transferred to the next generation and segregated from the transgenes. Rationale Rice was one of the three most important food crops in the world (along with maize and wheat) and was the most economically important food crop. It is the main food crop that has been providing energy for the living activities of billions of people around the world today. Rice was one of the crops that need huge amounts of water (2500 L) for 1 kg production (Bouman.
Wet rice farming uses up to 80% of the total irrigation water in agriculture (Bouman. According to the Ministry of Natural Resources and Environment (2007) forecast, due to the effects of climate change, Vietnam will experience a severe water shortage in the next 50 years. Drought was one of the causes leading to severe rice yield declines (Hu et al. In Vietnam, the rice area in 2021 was estimated at 7.24 million hectares, down 38.3 thousand hectares compared to 2020 due to the effects of drought, salinization, and change of use purposes on rice land, so the sown area reduced planting reduces the whole crop's output.
Abiotic stress factors such as temperature, drought, and salinity have been seriously affecting the yield and quality of rice. The rice yield in the field with saline intrusion was only 52.45% compared to the field without saline intrusion (Kumar et al. In the year 2050, the world's population was projected to reach 9.1 billion, increasing by 34%. According to the Food and Agriculture Organization of the United Nations (FAO, 2018), the global demand for agricultural products will increase by about 70% by 2050.
In order to feed the projected world population, annual cereal production need to increase from 2.1 billion today to about 3. Based on these, improving plant resistance to biotic and abiotic stresses and increasing crop yield were major concerns in current agricultural research (Delorge et al. In the past decade, genome editing technologies have gained remarkable progress in developing climate-resilient and tolerant rice varieties that can withstand adverse conditions to sustain growth. The techniques 2 of random mutagenesis, naturally occurring mutations, and classical breeding techniques have been gradually replaced by new precision genome editing techniques because these methods take time to collect the individuals with the desired shape.
The new techniques have become important tools for plant molecular breeding plant science and plant science (Endo et al. Gene editing technology based on editing systems such as zinc finger nucleases (ZFNs), transcription activator–like effector nucleases (TALENs), Clustered Regularly Interspaced Short Palindromic Repeats system/ CRISPR- associated 9 (CRISPR/Cas9) was being perfected and applied on many plants as well as rice plants. Compared with previously developed gene editing tools ZFNs (Shukla et al., 2009) and TALENs (Christian et al., 2010), CRISPR/Cas9 was simpler in design, more efficient and it can edit multiple target genes simultaneously (Zhao et al. Therefore, CRISPR/Cas9 was a real potential system to modify genomic DNA in many crops including rice.
In addition, through time, to survive under various stress conditions, plants have evolved complex mechanisms to perceive external signals and respond to changing environmental conditions. These mechanisms include stress perception, signal transduction, transcriptional activation of stress-responsive target genes, and stress-related synthesis of proteins or other molecules that help plants cope with adverse conditions via biochemical and physiological manifestations (Zhu JK. Recently, the mechanisms of environmental stress responses have been focused more by studies that can be genetically engineered to develop stress- tolerant crops. In previous studies, the Oryza sativa Delayed Seed Germination 1 (OsDSG1), a RING finger E3 ligase gene which regulates importance in the control of seed germination and responses following exposure to multiple stresses (Park et al.
In the osdsg1 mutant which silenced by RNAi are drought and salinity tolerance, due to the transcript levels of ABA signaling and responsive genes were significantly increased. However, there are still some limitations such as the delayed-germination phenotype and mutant plants were shorter than wide- type plants. 3 Therefore, creating mutations on the DSG1 by CRISPR-Cas 9 system should be a potential strategy to eliminate previous limitations. Using Agrobacterium mediated method, the research group at the Institude of Biotechnology has created different transgenic lines carrying CRISPR/Cas9 construct to targeted inducing mutation of the DSG1 promoter.
In this study, we conducted “Characterization of mutations in the OsDSG1 promoter of rice lines induced by the CRISPR-Cas 9 system” 1. Aim of the study Analyzing and evaluating the heritability of mutations on the promoter of the DSG1 gene in gene-edited rice lines using CRISPR/Cas9 technology 1. Objectives - To identify and characterize CRISPR/Cas9 induced mutation of the OsDSG1 promoter mutations from the T0 transgenic rice - To assess inheritance and segregation of the OsDSG1 promoter mutations at the T1 generation 4 Chapter II. General introduction of rice (Oryza sativa L.