VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY GRADUATION THESIS TITLE: DESIGN T-DNA VECTOR EXPRESSING OsNRAMP7 GENE INVOLVED IN METAL ACCUMULATION IN RICE VARIETY TBR225 Hanoi, 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY ------- ------- GRADUATION THESIS DESIGN T-DNA VECTOR EXPRESSING OsNRAMP7 GENE INVOLVED IN METAL ACCUMULATION IN RICE VARIETY TBR225 Student : Nguyen Hoang Minh Class : K63CNSHE Major : Biotechnology Supervisor : Ninh Thi Thao, Ph. HANOI, 2022 COMMITMENT 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, December 2022 Student Nguyen Hoang Minh 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 Dr. Ninh Thi Thao 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 BCs. 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, December 2022 Student Nguyen Hoang Minh ii CONTENTS CONTENTS.
iii LIST OF ABREVIATIONS. vi LIST OF FIGURES. Aim, objectives and meaning of research. Aim of the research.
Meaning of the topic. OVERVIEWS OF LITERATURE. Micronutrients in rice. Effect of micronutrient on plant growth.
Effect of micronutrient on human health. Researches on improving micronutrient content in rice. NRAMP protein in plants. Functional characteristics of NRAMP.
Regulation of NRAMP gene expression in plants. Researches on breeding rice varieties with high zinc content. Molecular mechanism of Zn accumulation in rice grain. Application of marker-assisted selection in high Zn rice breeding.
Plant transformation and plant gene expressing vector systems. General introduction of plant transformation. Methods of indirect plant transformation through A. Vector systems for plant transformation through A.
Overview about TBR225 rice variety. RESEARCH MATERIALS AND METHODS. Time and place of study. Equipments and chemicals.
General molecular biology techniques. Isolation of TBR225 OsNRAMP7. Cloning the OsNRAMP7-TBR into the vector pJET 1. Design of T-DNA vector expressing OsNRAMP7-TBR.
RESULTS AND DISCUSSION. Isolation of TBR225 OsNRAMP7 by PCR. Cloning and sequencing of the OsNRAMP7-TBR. Cloning OsNRAMP7-TBR.
Validation of the recombinant plasmid pJET/OsNRAMP7-TBR. Sequencing OsNRAMP7-TBR. Design of plant transformation vector overexpressing OsNRAMP7-TBR. Insertion of OsNRAMP7-TBR into vector pCAM-Ubi.
Validation of the recombinant vector pCAM-Ubi/OsNRAMP7-TBR. tumefaciens strain EHA105 carrying the vector pCAM-Ubi/OsNRAMP7-TBR. CONCLUSSIONS AND SUGGESTIONS. 44 v LIST OF ABREVIATIONS A.
tumefaciens Agrobacterium tumefaciens Bp Base pair CTAB Cetyltrimethylammonium bromide DNA Deoxy ribonucleotide Acid dNTP Deoxynucleotide triphosphate E. coli Escherichia coli EtBr Ethidium bromide Fe Iron Kb Kilobase Mn Mangan Nu Nucleotide NRAMP Natural resistance-associated macrophage protein NRAMP7-TBR TBR225 OsNRAMP7 PCR Polymerase chain reaction TAE Tris acetate T–DNA Transfer–DNA Ti–plasmid Tumor inducing plasmid Zn Zinc vi LIST OF TABLES Table 3.1: Sequence of oligonucleotides used in the research. 22 vii LIST OF FIGURES Figure 3.1: Diagram of pCAM-Ubi/OsNRAMP7-TBR vector design.1: Isolation of OsNRAMP7-TBR from cDNA.2: Structure diagram of pJET1.3: Transformation of the pJET/OsNRAMP7-TBR into E.4: Colony PCR of E. coli cells transformed pJET/OsNRAMP7-TBR.5: Validation of pJET/OsNRAMP7-TBR by PCR.6: Validation of pJET/OsNRAMP7-TBR by restriction enzymes.7: Sequencing TBR225 OsNRAMP7.8: Digestion of pJET/OsNRAMP7-TBR and pCAM-Ubi.9: Transformation of pCAM-Ubi/OsNRAMP7-TBR into E.10: Colony PCR of E.
coli transformed pCAM-Ubi/OsRAMP7-TBR.11: Validation of pCAM-Ubi/OsNRAMP7-TBR by PCR.12: Validation of pCAM-Ubi/OsNRAMP7-TBR by RE.13: Sequencing of pCAM-Ubi/OsNRAMP7-TBR.14: Colony-directed PCR products from A. 42 viii ABSTRACT Micronutrients are necessary for plant growth and development. The Natural resistance-associated macrophage protein (NRAMP) family is related to the accumulation of micronutrients in plants. In rice, there were eight genes belonging to the OsNRAMP family, of which OsNRAMP1, OsNRAMP4, OsNRAMP5, OsNRAMP6, and OsNRAMP8 were reported to transport metals such as iron, zinc, cadmium.
Recently, AtNRAMP5, closely related to OsNRAMP7, was identified as that function in Zn uptake in Arabidopsis. This study aims to construct the gene expression cassette for generating OsNRAMP7- overexpressing transgenic rice plants.6 kb of coding sequence of OsNRAMP7 was isolated from the total cDNA of the major rice variety TBR225 and cloned into the pJET2. The isolated gene fragment is 98.3% similar to the OsNRAMP7 sequence published on the GenBank (code XM_015762723. The coding sequence of OsNRAMP7 was inserted into the BamHI site on the binary vector carrying the gene expression construct driven by the constitutive promoter Ubiquitin.
The recombinant vector was validated by PCR, restriction enzyme, and DNA sequencing analysis. This study is a premise for generating transgenic rice lines overexpressing OsNRAMP7 to identify the function of the target gene.1 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. The NRAMP proteins have been found to transport metal ions such as Zn2+, Mn2+, Fe2+, Cd2+, and others in plants. Expression studies indicated 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 transpoter responsible for Fe, Mn and Cd, whereas OsNRAMP4 was identified as a transpoter for Al. However, despite 1 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. From the above facts, we studied this project "Designing T-DNA structure expressing OsNRAMP7 gene related to metal accumulation in rice" to generate the materials for functional study of OsRMAMP7 gene.
Aim, objectives and meaning of research 1. Aim of the research Design of T-DNA structure overexpressing OsRMAMP7 gene involved in metal accumulation in Vietnamese rice variety TBR225. Objectives - Isolation of the OsNRAMP7 gene from rice variety TBR225 (OsNRAMP7- TBR). - Cloning the OsNRAMP7- TBR gene.
- Design of T-DNA vector expressing OsNRAMP7-TBR. Meaning of the topic Generating the materials for studying the function of the OsNRAMP7 gene in the major rice variety TBR225. OVERVIEWS OF LITERATURE 2. Micronutrients in rice 2.
General introduction Rice makes up an important part of the daily diet, especially in developing countries in Asia, providing 50–80% of daily calories, protein, minerals and vitamins, etc. The annual global rice production of 700 million tons was produced on an area of 158 million hectares in 2017. However, rice production must increase by 8-10 million tons per year and there is a strong incentive to raise prices. nutritional value with many essential vitamins and minerals to meet the food and nutritional needs of the population.
Micronutrients are essential for human and animal health in addition to being required for plant growth and development. The idea of latent hunger (a lack of some vitamins and micronutrients while consuming enough calories) has been developed explicitly in the last two decades (Bashir et al. For both plants and animals to grow and develop normally, trace elements like Fe and Zn are crucial. Anemia is caused by a fe deficiency, which has also been linked to pathogenic effects.
Growth retardation, hypogonadism, immune system problems, and cognitive decline are all brought on by Zn deficiency. One of the most prevalent micronutrient deficiencies in humans, Fe and Zn deficiency affects two billion people and results in more than 0.8 million fatalities per year (Bashir et al. Humans should consume 15 mg of both Fe and Zn daily. However, rice-based diets, which are popular among Asian populations, rarely meet this need.
When compared to the recommended dietary intake of Fe for humans, which is 10-15 mg, polished rice only has an average of 2 mg Fe/kg and 12 mg Zn/kg of rice; this is despite the fact that infants, children, and adolescents are growing quickly; pregnant women need enough Fe for 3 themselves and their growing fetus; this is particularly true toward the end of pregnancy, when the baby is growing most rapidly; and menstruating girls and women need more Fe due to blood loss. More than 300 enzymes involved in the metabolism of carbohydrates, lipids, proteins, and nucleic acids use zinc as a cofactor, which highlights the significance of zinc for healthy growth and development in both plants and animals. Effect of micronutrient on plant growth Plants require micronutrients such as Fe, Zn, and Mn. Fe is involved in a variety of cellular processes, including respiration, chlorophyll biosynthesis, and photosynthetic electron transport.
The most obvious symptom of Fe deficiency is low chlorophyll (chlorophyll) content in young leaves (Marschner, 1995). Fe deficiency appears to also cause oxidative stress (Tewari et al., 2005; Bashir et al. The function of chloroplasts and mitochondria is dependent on Fe. The mitochondrial Fe transporter transports Fe to mitochondria (Bashir et al.
Additional Fe can be redirected to vacuoles (Kim et al., 2006; Zhang et al. Vacuolar Fe transporters 1 (OsVIT1) and OsVIT2 are thought to be important subcellular Fe transporters in rice (Zhang et al. Zn is involved in a variety of cellular processes (Ishimaru et al. Zn is essential for the metabolism of proteins, nucleic acids, carbohydrates, and lipids (Rhodes and Klug, 1993; Vallee and Falchuk, 1993).
Zn absorption must be precisely calibrated to ensure that the proper amount of Zn is present at all times (Ishimaru et al. In plants, Zn deficiency causes starch accumulation and inactive RNases, implying that the availability of Zn in the cell regulates RNA degradation (Suzuki et al. Mn functions as a cofactor or activator of enzymes with various functional groups and activities. Crop yields are significantly reduced in Mn-deficient crops due to low temperature stress and pathogen infection (Marschner, 1995; Hebbern et al.
It is thought that Mn transport shares an entry pathway with Fe and Cadmium (Cd).