VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY UNDERGRADUATE THESIS TITLE CONSTRUCTION OF CRISPR/CAS9 VECTOR FOR SILENCING CIF1 GENE OF TOMATO HANOI – 2021 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY UNDERGRADUATE THESIS TITLE CONSTRUCTION OF CRISPR/CAS9 VECTOR FOR SILENCING CIF1 GENE OF TOMATO STUDENT: Nguyen Thi Bich Ngoc MAJOR: Biotechnology SUPERVISORS: Huynh Thi Thu Hue, PhD. Institue of Genome Research, VAST Tran Thi Hong Hanh, MSc. Vietnam National University of Agriculture HANOI – 2021 COMMITMENT I hereby undertake that this is my research project under the scientific guidance of PhD. Huynh Thi Thu Hue and MSc.TranThi Hong Hanh.
The results and data in this thesis have not been published by anyone in any way. This is part of the findings of the Genome Biodiversity Laboratory - Institute of Genome Research. 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 Student Nguyen Thi Bich Ngoc i ACKNOWLEDGMENTS First and foremost, I would like to express my deep thanks to PhD. Huynh Thi Thu Hue, Assoc. Prof Nguyen Xuan Canh, MSc. Tran Thi Hong Hanh, who helped me with my graduation thesis.
They taught me wholeheartedly as well as always created the most favorable conditions for me during the experiments to complete this graduation thesis. I would like to express my sincere thanks to the Vietnam National University of Agriculture, the Faculty Board and the teachers in the Faculty of Biotechnology for creating an interesting learning environment and providing me with invaluable knowledge. as valuable experiences during the past 4 years. During the course of the thesis at the Laboratory of Genome Biodiversity, Institute of Genome Research, Vietnam Academy of Science and Technology (VAST) with the enthusiasm of PhD.
Huynh Thi Thu Hue and staff of laboratory helped me complete my thesis and draw me a lot of experience. They are really dedicated to the profession, devote their best and have inspired me great inspiration in scientific research. Once again, I would like to express my deep thanks to the people who helped me to complete my thesis, and the leadership of the Institute of Genome Research for creating conditions for me to work here. In the end, it is impossible not to mention my parents.
I would like to express my deep thanks to my parents who always supported and encouraged me throughout the thesis making process and life in general. Once again, I would like to send my sincere thanks to everyone! Student Nguyen Thi Bich Ngoc ii TABLE OF CONTENTS ACKNOWLEDGMENTS. ii TABLE OF CONTENTS. iii LIST OF TABLES.
v LIST OF FIGURES. vi LIST OF ABBREVIATIONS. General introduction of Tomato. Tiny-Tim Tomato.
Studies of tomatoes. Researches on disease resistance and yield enhancement. Researches on fruit quality. CIF1 gene in plants.
Biological techniques for gene editing technology. ZFNs and TALENs. MATERIALS AND METHODS. Time and place of study.
Leaf samples of Tiny-Tom tomato varieties. Vector and primer. Strains of bacteria: E. Chemicals and reagents.
Genome DNA extraction. Specific gRNA designing for CIF1 gene. Ligation gRNA into pRGEB31 vectors. Plasmid transformation into E.coli by heat shock.
PCR technique check. Plasmid transformation into A. tumefaciens cells by electrical impulses. Sanger DNA sequencing.
RESULTS AND DISCUSSION. Total DNA extraction of Tiny-Tom tomato. Exon 1 region amplifying and sequencing. gRNA specific designing to CIF1 gene .coli strain with gRNA-CRISPR/Cas9 vector.
Colonies selection containing recombinant plasmids by PCR. Sequencing result of CRISPR/CAS9-CIF1. tumefaciens strains with gRNA-CRISPR/Cas9 vector. CONCLUSION AND RECOMMENDATIONS.
36 iv LIST OF TABLES Table 3. List of primers. Composition of PCR reaction. The composition reaction denaturation and renaturation created SlCIF1-G1 and SlCIF1-G2.
Components that cut- ligation the vector and heating cycle. Components PCR multiply the CIF1-G1 fragment. Components PCR multiply the CIF1-G2 fragment. The sequence of gRNAs.
30 v LIST OF FIGURES Figure 1. Tiny-Tim Tomatoes. CIF1 gene in Tomato. Mechanism of CRISPR/Cas9 systems.
Schematic structure of pRGEB31 vector. Schematic of duplication and insertion of gRNA into pRGEB31 vector. Total DNA of leaf samples. The optimal PCR reaction for exon 1 of SlCIF1 gene.
Purified PCR products and sequence of exon 1 SlCIF1. PCR checks for colonies containing structures pRGEB31-CIF1G1, pRGEB31-CIF1G2. Sequencing gRNA on pRGEB31-CIF1-G2 vector number 1, 2, 4, 5. Plasmid extraction and plasmid contains pRGEB31- CIF1-G2 structures in PCR reaction.
33 vi LIST OF ABBREVIATIONS Abbreviations Definitions AAT Alpha-1 Antitrypsin bp basepair Cas CRISPR-associated CDS Coding sequence CIF1 Cell wall inhibitor of β-fructosidase CIN Cytoplasmid invertases CWI/CWIN Cell wall invertases CRISPR Clustered Regularly Interspaced Short Palindromic Repeats crRNA CRISPR RNA DBS DNA double-strand break DNA Deoxyribonucleic Acid FIX Factor IX gRNA Guide RNA INVINH Invertase inhibitors iPS Induced pluripotent stem cells MG Mature green PAM Protospacer adjacent motif PB PiggyBac vector PCR Polymerase Chain Reaction R Rip rAAV Recombinant adeno-associated virus RIN Ripening inhibator RNA Ribonucleic Acid RNAi RNA interference Ta Annealing temperature TALEN Transcription ativator-like effector nucleases vii Tm Melting temperature tracrRNA Trans-activating CRISPR RNA VI/VIN Vacuolar inhibitor of β-fructosidase ZFNs Zinc-finger nucleases kb Kilobase mM Milimolar min Minute ng Nanogram NCBI National Center for Biotechnology Information nm Nanometer rpm Revolutions per minute µL Microlitre viii ABSTRACT Solanum lycopersicum tomatoes is a nutrient-dense food that contains many secondary compounds with great health benefits. Tomato fruit production has a high sugar content through the regulation and breakdown of sucrose. Cell wall invertase (CWI) hydrolyzes sucrose into monosaccarit and transports it into cytoplasma so that sugar content of tomato is regulated by CWI. But the invertase is inhibited by a protein encoded by CIF1 gene.
So, inactivation of the CIF1 gene will show its effect on sugar synthesis in tomatoes. Nowadays, the CRISPR / Cas9 system is increasingly used in the editing of desired genes in plant subjects. In this study, gRNAs which target on tomato CIF1 gene were designed and inserted into CRISPR/Cas9 vectors. In the research, created DH10B E.coli strains carrying pRGEB31-CIF1-G2 vectors contains CRISPR/Cas9 expression structures towards CIF1 geneof tomato.
In addition, a strain of A. tumefaciens harboring pRGEB31-CIF1-G2 vector carrying CRISPR/Cas9 expression system towards CIF1 gene in tomato were successfully created. The strain of A. tumefaciens harboring pRGEB31- CIF1-G2 plasmid will be used to develop transgenic tomato plants from Tiny-Tim variety.
Therefore, it may help to facilitate the gene edditing in tomatoes as well as the application of this technique to other crops. Introduction Tomato is a crop that has a lot of nutritional value to humans. Furthermore, this is a fruit tree with a large demand for production and consumption, which is increasing in agriculture. Therefore, there have been number of studies on tomatoes conducted for many decades to increase the understanding of tomato plants, in order to meet the human needs to improve fruit yield and quality.
During plant growth, hormones play an important role in regulating the proper development. Through each stage of plant development with growth stimulating effects such as Auxin, Gibberellin, Cytokinin or growth inhibitors such as Abscisic acid, Ethylene. Besides, the sugar ingredient plays a key role in producing the delicious taste of ripe tomatoes. The sugar content of ripe fruit is roughly 50% of the dry weight.
The sugar content in the fruit increases gradually from the mature green stage to the red ripe stage (Davies et al. This content of ripe fruit is mainly glucose and fructose in equal proportions. In addition, the sucrose (disaccharide) content is negligible (less than 0.1%), which is transported from the leaves to the fruit. Thus, the sweetness of ripe tomatoes is chiefly due to the composition of monosaccharide (Davies, 1966; Davies et al.
Subsequently, a system for a genetic engineering vector was designed to inactivate the CIF1 gene, which codes for a CWI inhibitor protein. This vector was conducted to evaluate the function of the CIF1 gene in the Tiny-Tim tomato plant. The advancement of molecular biology techniques gives scientists the tools to study the effects of genes in cells. Successive gene editing systems help overcome the limitations and enhance the efficiency, consist of ZFNs, TALENs.
Recently, the CRISPR /Cas system, with advantages such as time and cost, has brought many in- depth studies in molecular biology field. Accordingly, we carried out the research project "Construction of CRISPR/Cas 9 vector for silencing CIF1 gene of tomato". In this study, the CRISPR / CAS9 gene editing system is applied to inactivate CIF1 gene. After, gRNA is designed specific for CIF1 gene and attach to pRGEB31 vector, this product is transformed into E.coli 1 strain DH10B.
The resulting product will be transformed into A. tumefaciens strain EHA105 to transfer genes into tomatoes. From that design, it can be applied for gene expression in Tiny-Tim tomato to better understand the function of the CIF1 gene. Objectives Purpose: Successfully generated gRNA to construct the CRISPR / Cas9 structure expressing vectors for silencing CIF1 gene.
Requirements: - High quality DNA extraction - Exon1 amplification of CIF1 gene. - Designed specific gRNA for CIF1 gene. - Transformation of competent E. tumefaciens cell with gRNA- CRISPR/Cas9 vector.
- Selection of colonies containing recombinant plasmids. General introduction of Tomato 2. Overview Tomato (Solanum lycopersicum) is one of the most nutritious vegetable crops in the world. Solanum lycopersicum (Figure 1.1) and its wild relatives (genus Solanum, part Lycopersicon) are native to western South America (Ecuador, Peru and Chile).
Wild tomatoes can still be found along the west coast of South America, in the Andes and on the Galapagos Islands. Although wild tomato diversity is concentrated in Peru (Rick, 1991b), genetic analysis of primitive cultivars has shown that the center of tomato diversity is Mexico. This suggests that tomato domestication may have occurred in Central America (Rick, 1991b). When the conquistadors came from Europe to the Americas, the cultivation of tomatoes was widespread.
It is likely that Europeans distributed tomatoes from the Americas to Europe and European colonies in the 16th century. Interestingly, the tomato was brought into the United States by European immigrants, not Mexico. Tomato (Solanum lycopersicum) Solanum lycopersicum is the most consumed vegetable in the world because it is the basic ingredient in many raw, cooked or processed foods. Besides, it has commercial value grown worldwide for local use or as an export crop.
In 2014, the 3 global tomato acreage was 5 million hectares with a production of 171 million tons, the two largest tomato producing countries were China and India (FAOSTAT, 2017). Tomatoes can be grown in a variety of geographical areas in fields or greenhouses, and fruit can be harvested manually or mechanically. Under certain conditions (e. regenerative pruning, weeding, irrigation, frost protection), this crop can be grown perennial or short-term, but commercially it is considered annual crops (Geisenberg and Stewart, 1986).
Although there are many types of greenhouse tomato systems available, the two main cropping systems are two crops a year and one crop a year. Its importance lies not only in profits, but also in the income generated in the local economy for farmers and farm workers (Villarreal, 1982; Coll-Hurtado and Godínez Calderón, 2003). Protected agriculture is a broad category of production modes that provides some degree of control over various environmental factors. This portfolio includes production technologies such as greenhouses, tunnels and covered fields (Nieves- García, van der Valk and Elings, 2011).
Although there are no quantitative data on world vegetable production in greenhouses, some calculations have been made. For example, in 2012, greenhouse vegetable production was about 81 million kg of which 40 million kg were tomatoes and 37 million kg were cucumbers. More specifically, in 2012, greenhouse tomato production in North America accounted for 52% of the market in Canada and 22% of the market in the United States (Farm Credit Canada, 2012). Today, tomatoes are one of the major vegetable crops in the world.