Characterization of mutations in the osdsg1 promoter of rice lines induced by the crisprcas9 system

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 Without Dr 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 i ACKNOWLEDGEMENTS ii LIST OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS vii ABSTRACT Chapter I INTRODUCTION 1.1 Rationale 1.2 Aim of the study 1.3 Requirement Chapter II LITERATURE REVIEW 2.1 General introduction of rice (Oryza sativa L.) 2.1.1 Rice (Oryza sativa L.) 2.1.2 Rice production in Viet Nam 2.2 Abiotic stress and abiotic stress resistance in rice 2.3 Overview of the Ring finger E3 ligases (OsDSG1) gene in rice 2.3.1 Gene OsDSG1, a Ring finger E3 ligases, to create stress-resistant transgenic plants 2.3.2 The role of DSG1 on growth and development in rice 12 2.3.3 The role of DSG1 in salinity and drought tolerance in rice 13 2.4 Promoter 14 2.5 Application of CRISPR/Cas9 in generating mutations on the DSG1 promoter 16 Chapter III MATERIALS AND METHODS 19 3.1 Materials and chemicals 19 3.1.1 Materials 19 3.1.2 Chemicals 20 3.1.3 Equipment 20 3.2 Method 21 3.2.1 Check the presence of editing system in transgenic T0 and T1 plants 21 3.2.1.1 Total DNA extraction 21 iii 3.2.1.2 Detection of transgenes in T0 and T1 plants based on Hygromycin 21 3.2.2 Analysis of rice lines T0 carrying mutations of the target gene 22 3.2.2.1 Mutation detection by analysis of DNA Heteroduplexes 22 3.2.2.2 Amplify DSG1 promoter region 22 3.2.2.3 Cloning of DSG1 promoter 23 3.2.2.5 Sequencing and analysis of sequence results 25 3.2.3 Inheritance of OsDSG1 promoter mutations at the T1 generation 25 Chapter IV RESULTS AND DISCUSSION 27 4.1 Analysis of DSG1 promoter mutation in T0 plants 27 4.1.1 Detect transgenes in T0 plants 27 4.1.2 Mutation detection of DSG1 promoter in T0 plants 28 4.1.3 Amplify DSG1 promoter region 30 4.1.4 Cloning of DSG1 promoter 31 4.1.5 Sequencing and analysis of sequence results in T0 generation 33 4.2 Inheritance of OsDSG1 promoter mutations at the T1 generation 34 4.2.1 Analysis of mutations in T1 plants 34 4.2.2 Sequencing and analysis of sequence results in T1 generation 37 4.2.3 Detect transgenes in T1 plants 38 4.3 Discussion 39 4.3.1 Mutations induced in the DSG1 promoter in the T0 plants 40 4.3.2 Inheritance of DSG1 promoter mutations at the T1 generation 40 Chaper V: CONCLUSIONS 42 5.1 Conclusions 42 5.2 Proposals 42 REFERENCES 43 iv LIST OF TABLES Order Name of table Page Table 3.1 Components of the PCR reaction 22 Table 3.2 Primers were used in PCR 23 Table 3.3 Primers were used in PCR 25 Table 3.4 Components of the PCR reaction 26 Table 4.1 Investigate the inheritance of mutant alleles in the T1 generation 37 v LIST OF FIGURES Order Name of figure Page Figure 2.1 Working model of ABA-signaling pathway in germinating rice seeds 12 Figure 2.2 Phenotypes of OsDSG1 13 Figure 2.3 Abiotic stress responses 14 Figure 2.4 Model of promoter showing core promoter, proximal promoter, and distal promoter regions (Carlos M et al., 2014) 16 Figure 3.1 Schematic representation of CRISPR/Cas9-mediated targeted mutagenesis in the rice OsDSG1 gene 19 Figure 4.1 Electrophoresis of PCR product of with Hpt F/R primers 27 Figure 4.2 Detection of mutations in OsDSG1 promoter in T0 plants using heteroduplex analysis 30 Figure 4.3 Electrophoresis of PCR products amplified from OsDSG1 promoter in T0 plants, using PrDSG1-F1667/R260 30 Figure 4.4 Electrophoresis of PCR products to determine the presence of OsDSG1 promoter in T0 plants after being transformed 33 Figure 4.5 Sequence alignment of DSG1 promoter alleles in T0 plants 34 Figure 4.6 PCR detects the mutation DSG1 promoter of the T1 progeny were derived from three T0 plants: 2.2, 2.3, 3.1 37 Figure 4.7 Cas9/gRNA-induced homozygous mutations in the DSG1 promoter found in T0 and T1 mutant population 38 Figure 4.8 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 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 costeffective 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 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 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 of 16 tested T1 plants carrying homozygous mutations and free of transgenes This result indicates that the CRISPR/Cas9mediated mutations in the DSG1 promoter were successfully transferred to the next generation and segregated from the transgenes M WT (+) 16 17 (-) 400 300 (G) M WT (+) 10 11 12 14 20 21 22 (-) 400 300 (H) M WT (+) (-) 300 200 (I) M WT (+) 10 11 12 14 16 17 20 21 22 (-) 300 200 (J) 36 Figure 4.4 Detection of mutations in DSG1 promoter in T1 plants by PCR Line 2.2 (A) using PrDSG1-F563/R260 primers (B) using PrDSG1-R861/R260 primers Line 2.3 (C), (D) using PrDSG1-F1667/R1311 primers (E), (F) using PrDSG1-F1667/F1122 primers Line 3.1 (G), (H) using PrDSG1- F1667/R1311 primers (I), (J) using PrDSG1F1667/F1122 primers M: marker 100bp, WT: wild-type plant , +: pHUE-DSG1 plasmid Table 4.1 Investigate the inheritance of mutant alleles in the T1 generation T1 Transgenic T1 plants lines Bialleic mutants Homozygous mutants (ab) Percentage of bialleic mutants (ab) aa bb Percentage of homozygous mutants Percentage of homozygous mutants (aa) (bb) 2.2 12 50% 16,67% 33.33% 2.3 14 3 57.14% 21.43% 21.43% 3.1 16 12 75% 6.25% 18.75% Note: Transgenic plants with addition or/and deletion one or more nucleotides mutation were denoted a and inversion mutation were denoted b 4.2.2 Sequencing and analysis of sequence results in T1 generation In the above analysis, we identified totally 16 individual induced mutations in three transgenic T1 lines Subsequently, 10 individual lines were subjected for Sanger sequencing The sequencing result (Fig 4.7) showed, that all mutations of T1 plants were inherited from the T0 lines We founded that 10 transgenic lines were homozygous mutations T0 plants: Alleles g1552 g1023 g407 ATG 2.2-b : +1 _+1