VIETNAM AGRICULTURE ACADEMY FACULTY OF ECONOMY AND RURAL DEVELOPMENT GRADUATION THESIS DESIGN OF A CRISPR/CAS9 SYSTEM TO OVEREXPRESS OsNRAMP7 IN RICE VARIETY TBR225 Hanoi, July 2022 VIETNAM AGRICULTURE ACADEMY FACULTY OF ECONOMY AND RURAL DEVELOPMENT ------- ------- GRADUATION THESIS TOPIC: DESIGN OF A CRISPR/CAS9 SYSTEM TO OVEREXPRESS OsNRAMP7 IN RICE VARIETY TBR225 Student : Ngo Thi Van Anh Class : K64CNSHE Major : Biotechnology Instructor : Dr. Nguyen Duy Phuong : Assoc. Dong Huy Gioi HANOI, 2022 GUARANTEE I hereby declare that this thesis is my research. The collected data are true and have never been used or published in any documents, dissertations and scientific works ever before.
I hereby declare that the cited information in this thesis has been made of the source, ensuring cited as prescribed. I bear full responsibility for these reassurances. Hanoi, July 2022 Student Ngo Thi Van Anh i ACKNOWLEDGEMENTS In order to complete this graduation thesis, in addition to my constant efforts, I have received a lot of attention and help from groups and individuals inside and outside the school. First and foremost, I would like to offer my deep gratitude to Assoc.
Dong Huy Gioi and teachers from Faculty of Biotechnology for imparting me critical and valuable knowledge during the study and training process at the Vietnam National University of Agriculture. I would like to express my respect and appreciation to my supervisors Dr. Nguyen Duy Phuong and the teachers at the Agricultural Genetics Institute (AGI) for having enthusiastically guided, advised and supported me throughout the process of learning and study; as well as Agricultural Genetics Institute (AGI) for creating favourable conditions to help me finished my thesis. I would like to express my gratitude to Mr.
Nguyen Anh Minh has supported me in the lab works and my friends have helped and encouraged me during practice time. Finally, I would like to send my sincerest thanks to my family and loved ones, who have supported and assisted me during practice time. Due to limited time and knowledge, the thesis inevitably has certain limitations and shortcomings. I would like to thank and appreciate the contributions from teachers, lecturers and students.
I sincerely thank! Hanoi, July 2022 Student Ngo Thi Van Anh ii LIST OF ABREVIATIONS A. tumefaciens Agrobacterium tumefaciens ABA Abscisic acid AP2 APETALA 2 Bp Base pair Cas CRISPR associated protein CRISPR Clustered regularly interspaced short palindromic repeats crRNA CRISPR RNA dNTP Deoxynucleotide triphosphate DSB DNA double-strand break E. coli Escherichia coli EtBr Ethidium Bromide gRNA Guide RNA HR Homologous recombination HPT Hygromycin phosphotransferase TBR225 Rice cultivar TBR225 Kb Kilobase NRAMP Natural Resistance Associated Macrophage Protein NHEJ Non-homologous end-joining Nu Nucleotide OsNRAMP7- The fragment of OsNRAMP7 gene was isolated from the TBR225 TBR225 rice cultivar PAM Protospacer adjacent motif PCR Polymerase chain reaction sgRNA single guide RNA TAL Transcription activator-like TALEN TAL effector nuclease tracrRNA trans-encoded CRISPR RNA ZFN Zinc-finger nuclease iii LIST OF TABLES Table 3. Sequence of oligonucleotides used in the research.
Characterization of TBR225 OsNRAMP7-editing sgRNAs. Identification of TBR225 OsNRAMP7-editing sgRNAs. Genomic DNA sequences homologous to designed OsNRAMP7-TBR225- targeting crRNA. 55 iv LIST OF FIGURES Figure 3.
Diagram of the OsNRAMP7 structure. Diagram of designing pENTR4-V3/sgRNA-NRAMP7. Diagram of designing pENTR4-V3/sgRNA-NRAMP7-35S. Diagram of designing pCas9/sgRNA-NRAMP7-35S vector.
Isolation of OsNRAMP7-TBR225 from genomic DNA. Sequencing the amplicon of OsNRAMP7-TBR225. Alignment of the OsNRAMP7. Location of crRNA on OsNRAMP7-TBR225.
Secondary structure of sgRNA targeting OsNRAMP7-TBR225. Insertion of crRNA12-OsNRAMP7 into vector pENTR4-V3. Validation of the plasmid pENTR4-V3/sgRNA-NRAMP7. Sequencing the recombinant vector pENTR4/sgRNA-NRAMP7.
Insertion of NRAMP7-35S into pENTR4-V3/sgRNA-NRAMP7. Validation of the plasmid pENTR4-V3/sgRNA-NRAMP7-35S. Sequencing the vector pENTR4/sgRNA-NRAMP7-35S. Insertion of sgRNA-NRAMP7-35S into pCas9.
Validation of the plasmid pCAS9/sgRNA-NRAMP7-35S. Transformation of pCAS9/sgRNA-NRAMP7-35s. 69 v ABSTRACT Micronutrients are not only necessary for plant growth and development, but also for human and animal health. Natural resistance-associated macrophage protein (NRAMP) family, that have been found to be related to the accumulation of micronutrients in plants.
In rice, there were 8 gene belong to OsNRAMP family of which OsNRAMP1, OsNRAMP4, OsNRAMP5, OsNRAMP6 and OsNRAMP8 were reported to transport metals such as iron, zinc, etc. Recent, AtNRAMP5 which was closely related to OsNRAMP7 was identified that function in Zn uptake in Arabidopsis. In order to overexpress OsNRAMP7 of rice variety TBR225, in this study, a part of the promoter and the Exon I of the gene was isolated with an approximate 800 bp in size for structural design of a gene editing construct. The isolated DNA fragment is 99.8 % similar to the homologous OsNRAMP7 sequence published on the GenBank (code CP012620).
Based on the sequencing analysis, the TBR225 OsNRAMP7-targeting sgRNA and TBR225 OsNRAMP7-homologous DNA template fragment containing CaMV35S promoter sequence were designed by the bioinformatic software. These fragments were, respectively, inserted into LguI and BamHI sites on pENTR4-V3 carrying the sgRNA expression construct which then subcloned into plant transformation vector pCas9. This research is the basis for functional identification of OsNRAMP7 and further creating new rice varieties with high micronutrient content by genetic engineering in Vietnam. vi CONTENTS ACKNOWLEDGEMENTS.
ii LIST OF ABREVIATIONS .iii LIST OF TABLES. iv LIST OF FIGURES. Aim, objectives and meaning of research. Aim of the research.
Meaning of the topic. OVERVIEWS OF LITERATURE. Plant metal transport proteins and the NRAMP family. Proteins involved in metal transport in plants.
The roles of NRAMP protein in plants. Research on breeding rice varieties with high zinc content. Selection of rice varieties with high Zn content. Molecular basis of Zn accumulation in rice grain.
Application of marker-assisted selection in high Zn rice breeding. CRISPR/Cas9 gene editing system. An overview of gene editing systems. An overview of CRISPR/Cas9.
Functional mechanism of CRISPR/Cas 9. Application of CRISPR/Cas9 in plant breeding. RESEARCH MATERIALS AND METHODS. Time and place of study.
Equipments and chemicals. General molecular biology techniques. Isolation of TBR225 OsNRAMP7 by PCR. Design of the sgRNA expression construct for targeting OsNRAMP7- TBR225.
Design of dual-sgRNA expression vector carrying DNA template for TBR225 OsNRAMP7 HDR-mediated editing. Design of plant transformation vector for editing TBR225 OsNRAMP7. RESULTS AND DISCUSSIONS. Isolation and sequencing analysis of the TBR225 OsNRAMP7.
Isolation of TBR225 OsNRAMP7. Sequencing analysis of the TBR225 OsNRAMP7. Design of sgRNA for OsNRAMP7-TBR225-targeting CRISPR/Cas9 complex 43 4. Construction of OsNRAMP7-TBR225-targeting sgRNA expression cassette.
Insertion of OsNRAMP7-TBR225-targeting crRNA into pENTR4-V3. Confirmation of the recombinant pENTR4-V3/sgRNA. Design of dual sgRNA expression vector carrying DNA template for TBR225 2 OsNRAMP7 HDR-mediated editing. Insertion of NRAMP7-35S into pENTR4-V3/sgRNA.
Confirmation of the recombinant pENTR4-V3/sgRNA-NRAMP7-35S. Design of plant transformation vector for editing TBR225 OsNRAMP7. tumerfaciens strain containing the recombinant vector pCas9/sgRNA-NRAMP7-35S. CONCLUSSIONS AND SUGGESTIONS.
Introduction Rice (Oryza sativa L.) is one of the major food crops of the world in general and Vietnam in particular, which affects the diet of at least 65% of the world's population. Rice is a staple food, popular because it's a rich source of energy, is gluten-free, easy to digest, low in fat, and packed with vitamins, minerals, and other nutrients. However, some key elements such as iron are often lost when the bran layer of unmilled brown rice is scrubbed away to produce white rice. In developing countries, fortifying rice grains after milling may not be a viable option.
In addition, soils in which these crops are grown may lack some essential minerals, or minerals are not available for uptake by plant roots. There are concerns about micronutrient deficiencies in the diet, especially in countries where rice is the staple food. The important role of rice has prompted scientists to research and develop new rice varieties, in order to contribute to improving the nutritional balance of rice, ensuring food quality and security. The research to improve the nutritional value of rice was started nearly 20 years ago, initially using farming methods, traditional breeding and hybridization, now applying genetic technology, including genome editing.
The NRAMP (natural resistance-associated macrophage protein) transporter protein family has seven members in rice, including OsNRAMP1, OsNRAMP2, OsNRAMP3, OsNRAMP4, OsNRAMP5, OsNRAMP6, and OsNRAMP7. NRAMP have been found to transport metal ions such as Zn2+, Mn2+, Fe2+, Cd2+, and others in plants. Expression studies in normal plant tissues indicate that while OsNRAMP1 is expressed primarily in roots, and OsNRAMP2 is primarily expressed in leaves, OsNRAMP3 is expressed in both tissues. Furthermore, OsNRAMP5 is the main 4 transpoter responsible for Fe, Mn and Cd, whereas OsNRAMP4 was identified as a transpoter for Al.
However, despite this, relatively little information about these transporters is available, especially information regarding OsNRAMP7. The close genetic relationship between OsNRAMP7 and the identified Zn transporter AtNRAMP4 in Arabidopsis suggested the role of OsNRAMP7 in Zn accumulation in rice. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 protein) system was discovered in bacteria and archaea and can degrade exogenous substrates. It has garnered a lot of interest in recent years as a effective gene-editing tool for gene mutation and transcriptional regulation in animals and crops.
This technique allow precise and efficient modification of genomic DNA in cells through non-homologous end joining (NHEJ) repair or homology-directed repair (HDR) mechanisms. While NHEJ-mediated gene editing induces ramdom modification, HDR-mediated gene editing results in precise gene knock-in or replacement. Therefore, it is considered as an ideal tool to replace the traditional gene transformation method to overexpression target genes. From the above facts, we will conduct the project "Design of a CRISPR/Cas9 system to overexpress OsNRAMP7 in rice variety TBR225" to create the research materials for the study on functional identification of OsNRAMP7 and further high nutritional rice breeding program in Vietnam.
Aim, objectives and meaning of research 1. Aim of the research Designing a CRISPR/Cas9 system targeting OsNRAMP7 for gene overexpression in rice variety TBR225. Objectives - Isolation and sequencing of the OsNRAMP7 gene/promoter involved in metal accumulation in rice variety TBR225. - Design of a dual expression vector containing TBR225 OsNRAMP7-editing sgRNA expression construct and DNA template cassette harboring the promoter 35S and TBR225 OsNRAMP7-homologous sequences.
- Design of a plant transformation CRISPR/Cas9 vector for overexpression of TBR225 OsNRAMP7. Meaning of the topic Developing research materials for the study on functional identification of TBR225 OsNRAMP7 and further breeding program of rice variety TBR225 with high micronutrient content.OVERVIEWS OF LITERATURE 2. Plant metal transport proteins and the NRAMP family 2. Proteins involved in metal transport in plants Plants require a variety of essential transition metals as micronutrients for normal growth and development.
These metals are required for most redox reactions, which are fundamental to cell function. Fe is an important component of haem proteins (such as cytochrome, catalase and Fe-S-containing proteins such as ferredoxin) and various enzymes. Cu is an integral part of several electron transport proteins in photosynthesis (such as plastocyanins) and respiration (such as cytochrome c oxidase) and participates in liquefaction (laccase), although Mn has low redox activity, also participates in photosynthesis (evolved O2, etc. Zinc is not involved in redox activity, but is an important structure and/or catalyst involved in many proteins and enzymes.
Other transition metals such as Ni and Mo are also micronutrients essential for plant function. Loss of one of these metals can lead to a variety of deficiency symptoms and impaired growth (Marschner et al. However, although these metals are essential, they can be toxic if present in excess, and the production of reactive oxygen species and the harmful effects of oxidation are of particular importance (Schützend et al. Therefore, their intracellular concentrations need to be carefully controlled, and thus plants and other organisms have many potential mechanisms for metal ion homeostasis and tolerance, including transmembrane transport (Clemens et al., 2001; Hall et al.
Therefore, for healthy plant growth and development, a wide range of transition metals must be absorbed from the soil, transported within the plant, and distributed and divided in various tissues and cells. It is clear that membrane transport systems are likely to play an important role in this situation.