Construction of crisprcas9 vector for silencing cif1 gene of tomato (khóa luận tốt nghiệp)

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 ....................................................................................... vii
ABSTRACT ................................................................................................................... ix
CHAPTER I. INTRODUCTION .................................................................................... 1
1.1.

Introduction .......................................................................................................... 1

1.2.

Objectives ............................................................................................................. 2

CHAPTER II. LITERATURE REVIEW ........................................................................ 3
2.1.

General introduction of Tomato ........................................................................... 3

2.1.1. Overview .............................................................................................................. 3
2.1.2. Taxonomy ............................................................................................................. 5
2.1.3. Plant characteristics .............................................................................................. 6
2.1.4. Tiny-Tim Tomato ................................................................................................. 7
2.2.

Studies of tomatoes............................................................................................... 8

2.2.1. Researches on disease resistance and yield enhancement .................................... 8
2.2.2. Researches on fruit quality ................................................................................... 9
2.3.

CIF1 gene in plants ............................................................................................ 10

2.4.

Biological techniques for gene editing technology ............................................ 12

2.4.1. ZFNs and TALENs............................................................................................. 12
2.4.2. CRISPR/Cas9 ..................................................................................................... 14
CHAPTER III. MATERIALS AND METHODS ......................................................... 17
3.1.

Time and place of study ..................................................................................... 17

3.2.

Materials ............................................................................................................. 17

3.2.1. Leaf samples of Tiny-Tom tomato varieties. ..................................................... 17
3.2.2. Vector and primer ............................................................................................... 17
3.2.3. Strains of bacteria: E.coli and A. tumefaciens ................................................... 18
3.2.4. Chemicals and reagents ...................................................................................... 18
iii


3.2.5. Equipment........................................................................................................... 19
3.3.

Methods .............................................................................................................. 20

3.3.1. Genome DNA extraction .................................................................................... 20
3.3.2. Specific gRNA designing for CIF1 gene ........................................................... 20
3.3.3. Ligation gRNA into pRGEB31 vectors.............................................................. 22

3.3.4. Plasmid transformation into E.coli by heat shock .............................................. 24
3.3.5. PCR technique check .......................................................................................... 24
3.3.6. Plasmid extraction .............................................................................................. 25
3.3.7. Plasmid transformation into A. tumefaciens cells by electrical impulses .......... 26
3.3.8. Sanger DNA sequencing .................................................................................... 26
CHAPTER IV. RESULTS AND DISCUSSION .......................................................... 27
4.1.

Total DNA extraction of Tiny-Tom tomato ....................................................... 27

4.2.

Exon 1 region amplifying and sequencing ......................................................... 27

4.3.

gRNA specific designing to CIF1 gene ............................................................. 29

4.4.

Transformation of E.coli strain with gRNA-CRISPR/Cas9 vector.................... 30

4.4.1. Colonies selection containing recombinant plasmids by PCR ........................... 30
4.4.2. Sequencing result of CRISPR/CAS9-CIF1 ........................................................ 31
4.5.

Transformation of A. tumefaciens strains with gRNA-CRISPR/Cas9 vector. .. 33

CHAPTER V. CONCLUSION AND RECOMMENDATIONS .................................. 35
5.1.

Conclusion .......................................................................................................... 35

5.2.

Recommendations .............................................................................................. 35

REFERENCES .............................................................................................................. 36

iv


LIST OF TABLES
Table 3.1. List of primers ............................................................................................ 18
Table 3.2. Equipments ................................................................................................. 19
Table 3.3. Composition of PCR reaction .................................................................... 21
Table 3.4. The composition reaction denaturation and renaturation created
SlCIF1-G1 and SlCIF1-G2 ........................................................................ 23
Table 3.5. Components that cut- ligation the vector and heating cycle ...................... 23
Table 3.6. Components PCR multiply the CIF1-G1 fragment ................................... 24
Table 3.7. Components PCR multiply the CIF1-G2 fragment ................................... 25
Table 4.1. The sequence of gRNAs............................................................................. 30

v


LIST OF FIGURES

Figure 1.1. Tomato (Solanum lycopersicum) .................................................................. 3
Figure 2.2. Tiny-Tim Tomatoes ...................................................................................... 8

Figure 2.3. CIF1 gene in Tomato .................................................................................. 12
Figure 2.4. Mechanism of CRISPR/Cas9 systems. ....................................................... 15
Figure 3.1. Schematic structure of pRGEB31 vector .................................................... 17
Figure 3.2. Thermal cycle .............................................................................................. 21
Figure 3.3. Schematic of duplication and insertion of gRNA into pRGEB31 vector ... 22
Figure 3.4. Thermal cycling .......................................................................................... 25
Figure 4.1. Total DNA of leaf samples ......................................................................... 27
Figure 4.2. The optimal PCR reaction for exon 1 of SlCIF1 gene. ............................... 28
Figure 4.3. Purified PCR products and sequence of exon 1 SlCIF1 ............................. 29
Figure 4.4. PCR checks for colonies containing structures pRGEB31-CIF1G1,
pRGEB31-CIF1G2 .................................................................................... 31
Figure 4.5. Sequencing gRNA on pRGEB31-CIF1-G2 vector number 1, 2, 4, 5. ....... 32
Figure 4.6. 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.

ix


CHAPTER I. INTRODUCTION
1.1. 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., 1975). 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., 1981). 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 indepth 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.
1.2. 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.coli and A. tumefaciens cell with gRNACRISPR/Cas9 vector.

-

Selection of colonies containing recombinant plasmids.

2


CHAPTER II. LITERATURE REVIEW
2.1. General introduction of Tomato
2.1.1. 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.

Figure 1.1. 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.g.
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 (NievesGarcía, 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. Currently,
more than 120 million tons of tomatoes are produced annually worldwide. (Seisuke
Kimura and Neelima Sinha; 2014).
In addition, commercially important tomatoes may differ in color, size and
shape (Vaughan and Geissler, 1997). Fruit contains a large amount of water, vitamins
and minerals, low amounts of protein and fat and some carbohydrates. It also contains
carotenes, such as lycopene (which gives the fruit its red color predominantly) and
beta-Carotene (which gives the fruit its orange color). Modern varieties of tomatoes
produce fruit that contains up to 3% sugar by weight of fresh fruit. It also contains
tomatine, an alkaloid with fungicidal properties. The tomatine concentration decreased
4


as the fruit ripened and the tomatine concentration contributed to the species'
classification.
Therefore, it can be useful in plant propagation of cultivated tomatoes (OECD,
2008; Spooner, Anderson and Jansen, 1993). Tomatoes are related to wild tomatoes
native to Peru, Ecuador and other parts of South America including the Galapagos
Islands. Central to its domestication and diversification is Mexico (Rick, 1978;
Jenkins, 1948; Peralta, Spooner and Knapp, 2008). Wild relatives of tomatoes and
intermediate forms (race or creoles) contain a rich genetic diversity and are an

important source of genetic material in crop improvement and conservation programs
(Sánchez- Peña et al., 2004). Tomato is one of the best cultivated dicotyledon plants at
the molecular level and has been used as a model species for genetic mapping studies
(e.g., resistance genes). plant diseases) and genetic engineering. It is also useful when
studying other plant characteristics such as fruit ripening, hormone function and
vitamin biosynthesis (Gebhardt et al., 1991; Chetelat and Ji, 2006; Ji and Scott, 2006).
2.1.2. Taxonomy
Solanum lycopersicum is classified according to the IAPT international
classification system:
Classification
Kingdom

Plantae

Division

Magnoliophyta

Class

Magnoliopsida

Order

Solanales

Family

Solanaceae

Genus
Species

Solanum
S. lycopersicum

The cultivated tomato is a member of the Solanum genus in the Solanaceae
family. The Solanaceae, often referred to as the ornamental family, also includes other
notable crops such as tobacco, chili peppers, potatoes and eggplant. The classification
of tomatoes has been the subject of much discussion and the diversity of the genus has
5


led to a reassessment of previous taxonomic treatments. The tomato was originally
named Solanum lycopersicum by Linnaeus in 1753; Lycopersicon lycopersicum (L.)
Karsten has also been used (Valdes and Gray, 1998). Miller (1768) in The Gardener's
Dictionary used Lycopersicon esculentum. Rick (1979) includes nine species in the
genus Lycopersicon. Tomatoes have been known as L. esculentum for a long time, but
recent research has shown that they are part of the Solanum genus and are now again
widely known as Solanum lycopersicum (Spooner, Anderson and Jansen, 1993; Bohs
and Olmstead, 1997; Olmstead and Palmer, 1997; Knapp, 2002; Spooner et al., 2005,
2003; Peralta et al., 2008).
The genus Solanum includes roughly 1500 species. The tomato genus
(Lycopersicon section, formerly recognized as the genus Lycopersicon) includes
cultivated tomatoes (Solanum lycopersicum) and 12 wild relatives, all of which are
indigenous to western South America. Tomatoes (Solanum lycopersicum) are derived
from two wild ancestral species, Solanum pimpinellifolium and Solanum cerasiforme.
Other wild species are useful in breeding for disease resistance, improving color and
desirable quality traits (Ranc et al., 2008). The 12 wild members of the genus
Lycopersicum exhibited high levels of phenotypic and genetic variability, including a

great diversity in the mating system and reproductive biology (see Hybrids and
Hybrids and Bedinger 2011). Peralta, Spooner and Knapp (2008) recognized 12 wild
tomato species; This is an increase of over 9 tomato species recognized by Rick,
Laterrot and Philouze (1990). Of these 12, informal groups of species were
implemented: 4 closely related green fruit species - S. arcanum, S. huaylasense, S.
peruvianum and S. corliomulleri - were classified as S. peruvianum sensu lato (sensu
lato to refer to a broad concept of a species). Another group of yellow to orange fruit
species has two species endemic to the Galapagos Islands: S. galapagense and S.
cheesmaniae.
2.1.3. Plant characteristics
The tomato is a perennial herbaceous plant, but it is usually grown as an annual
even when persisted every two years. Tomatoes are grown in tropical and temperate
climates on open fields or in greenhouses in temperate climates. Greenhouses are often
6


used for large-scale production. In warm climates with a light intensity suitable for
growth, approximately 45 days are needed from germination to fruit set and 90-100
days for fruit to begin ripening (Nuez, 2001). The end use of a crop, whether it is a
processing market or a fresh market, determines the variety to be sown, when it is
harvested and the harvest processes, be it manual or mechanical. (Nuez, 2001).
Plant growth habits range from indeterminate to indeterminate and can reach
up to 3 meters (m) in height. The main root can be several meters long. The stems are
angular and covered with hairy trichomes and glands that provide a characteristic odor.
The leaves are arranged alternately on stems with a 137.5° phyllotaxy. The leaves are
shaped from lobed to compound, with needle-shaped segments. The cypress leaves
usually consist of five to nine leaflets. The leaflet is small and has a dent. All leaves
are covered with undercoat, trichomes.
Tomatoes are spherical or egg-shaped. The fruit exhibits all of the common
features of the berries; a simple nutty fruit that wraps its seeds in the pulp. The outer

shell is a thin and fleshy tissue that includes the rest of the fruit as well as the placenta.
The color of the fruit is derived from the cells in the flesh tissue. The tomato fruit can
be two or more seeds. Between 50 and 200 particles are located inside the positioning
chambers and wrapped in a gelatinous film. Medium, the seeds are small (5 x 4 x 2
mm) and shaped like a lentil. The seed contains the embryo and endosperm and is
covered with a firm seed coat called the testes. Fruit development takes seven to nine
weeks after fertilization. Many of the end uses of tomatoes, as well as food and feed
safety considerations, include composition of important food and feed nutrients,
antinutrients, and allergies and toxic substances, detailed in the "OECD Consensus
Document on ingredient considerations for new varieties of tomatoes" (OECD, 2008).
2.1.4. Tiny-Tim Tomato
Tiny-Tim tomatoes are easy to grow and produce unique dwarf plants that grow
very well in small containers, windows, hanging baskets and small garden spaces. As a
potted plant, Purple Tomato seeds can be planted at any time of the year and are midseason fruit trees.

7


A great centerpiece and a point of interest in any garden, Tiny cherry trees yield
small bright red / sweet cherry tomatoes about 2cm (1") in diameter. Perfect snack
salad or tomato. Plant conditions that will produce astonishing yield of miniature fruit
that is usually 3: 1 higher than a tree.
This small, neat, definite tree has dark green leaves that typically grow to 20-40
cm (8-16 ") tall and 17 cm (6 1/2") in diameter. Plant in full sun to maintain a habit of
dwarfism. Tiny Tomato Plant Tim Heirloom will thrive under less sunlight than other
varieties, however fruit yield will be less and grow larger and less neat.
The tiny cherry tomato variety Tim Heirloom was introduced by the University
of New Hampshire in 1945. Purple tomato plant is resistant or resistant to the
following tomato diseases: Alternaria stem cancer and Stemphylium disease.

Figure 2.2. Tiny-Tim Tomatoes ()
2.2. Studies of tomatoes
2.2.1. Researches on disease resistance and yield enhancement
In Vietnam as well as in the world, tomatoes play an important role in
agricultural production and increase income on cultivated area. There are some
temperate climates that are suitable for growing tomatoes, but the area is limited,
others have a large area, but the climate is seasonal so only one crop can be grown. In
addition, the yield and quality of tomatoes in addition to depending on varieties also
depends on soil conditions and pests.

8


In particular, with the hot and humid climate of Vietnam and many other
countries, it is easy to develop microbiological diseases such as Pseudomonas
solanacearum, causing plants to wilt very quickly and cause death. morning blight or
slow wilt disease caused by Phytophthora infestans that causes leaf and fruit rot, and
Colletotrichum phomoides anthracnose often damages ripe fruit. That poses challenges
for the agricultural sector in the prevention, treatment and creation of clean and
disease-resistant varieties.
Therefore, scientists have focused on improving the productivity of tomato
plants by creating safe, disease-resistant and heat-resistant tomato varieties to expand
cultivation time as well as cultivated area. effects in climates unsuitable for tomato
growth.
There have been many scientists researching and creating transgenic tomato
plants, carrying disease-resistant and pest-resistant genes such as: Production of
transgenic tomato plants expressing Cry 2Ab gene for the control of some
lepidopterous insects endemic in Egypt (M.Saker et al., 2011); Transgenic tomato
plants expressing the Arabidopsis NPR1 gene display enhanced resistance to a
spectrum of fungal and bacterial diseases (Wan-Chi Lin et al., 2004); Genomics of

Fungal Disease Resistance in Tomato (Dilip R. Panthee et al., 2010);…
2.2.2. Researches on fruit quality
The tomato is both an important horticultural crop and an established model for
the development of fleshy fruits. Sweetness is one of the most important properties for
maturity and quality of ripe fruit. In the past, due to the lack of information regarding
the biological mechanisms controlling fruit sweetness, breeders mainly relied on
sensory evaluation to develop varieties with high sugar content. Recently, the
significant progress in understanding the pathophysiology of tomato fruit sugar
metabolism and functional genome has brought hope for improved fruit quality
(Matsukura, 2016).
On tomatoes, the sugar component plays an important role in creating the
delicious taste of ripe tomatoes. The sugar in ripe tomatoes accounts for about 50% of
the dry weight. The cumulative sugar content in the fruit increases gradually from the
9


mature green (MG) stage to the red ripe (R) stage (Davies et al., 1975). The sugar
content of ripe fruit is mainly glucose and fructose in equal proportions, while the
succrose double sugar content is negligible (less than 0.1%), although succrose is the
form of Sugar is transported from the leaves to the fruit. Therefore, the sweetness of
ripe tomatoes is mainly due to the composition of simple sugars (Davies, 1966; Davies
et al., 1981). In tomatoes, the glucose level was almost negligible from the MG to R
stage in both the pulp and juices, while the fructose levels increased markedly during
this time. The general trend is that fruit accumulating sugars gradually increases from
green to yellow stage and decreases slightly at ripening stage (Davies et al., 1975).
Investigations in cell wall inactivation by using RNAi obtained tomato plants
with a longer lifespan, larger seeds as well as 70% higher sugar content than the
control plants at the time. Fruits are 10 days old and lead to increased activity of
invertases from 40 to 65% in leaves (Jin et al., 2009). However, the RNAi approach is
a traditional gene transfer method and can lead to undesirable results for transgenic

plants after transgenic loci are forced to retain through breeding, selective (Younis et
al., 2014). Up to now, according to information exploitation around the world, we
found that there is no publication related to inactivation of INVINH genes through
CRISPR / Cas9 method. Therefore, with CRISPR / Cas9 technology applied in this
study, tomato plants with improved sweetness under modern methods will be created.
2.3. CIF1 gene in plants
The sugar content of tomatoes is regulated by Acid Cell wall invertase (CWI),
which hydrolyzes sucrose into monosaccarit and transports it into the cytoplasm. The
gene CIF1 encodes for a protein that inhibits CWI and enhances expression by a
transcription regulator of ripening inhibitor (RIN).
The sugar content of tomatoes is regulated by two types of invertase (1)
Invertase acid mosaic on the cell wall (Cell wall invertase - CWI), which hydrolyzes
sucrose into simple sugars and transport into the cell. cytoplasm and (2) vacuole
invertase (Vacuole invertase-VI) have a role in maintaining the ratio of sucrose /
hexose in the cytoplasm. (Itai et al., 2015; Qin et al., 2016). Invertases will be
inhibited by the invertase inhibitors (INVINH) through the post-translation regulatory
10


mechanism, the INVINHs will interact with the invertase to create inactivated
complexes. The expression of INVINHs is regulated by abcisic acid and is directly
related to the tolerance reactions of tomatoes to cold weather (Rausch et al., 2004; Jin
et al., 2009). Currently, researches in the world have isolated 3 genes coding for
INVINH cells, including 1 gene encoding for VI inhibitory protein, VIF, and 2 genes
coding for CWI inhibiting proteins, CIF1 and CIF2. In tomatoes, VIF expression was
inhibited by a synthesized ripening inhibitor (RIN) transcription regulator while CIF1
and CIF2 were enhanced by this factor (Qin et al., 2016).
In higher plants, sucrose, which is the final product of photosynthesis, is
transported from the source to the heterotrophic basins, leading to the release of carbon
and energy (Amor et al., 1995). Invertase is the basic foundation for adjusting phloem

unloading and setting the sink strength; and plays an important role in the breakdown
of sucrose. The identified invertases have been grouped into acidic invertases,
including CWIN and vacuole invertases (VIN), and cytoplasmic invertases (CIN),
which are predicted based on the optimal and local pH. subcellular chemistry (Sturm,
1999; Ruan et al., 2010). The ripe fruit of the tomato plant mainly accumulates glucose
and fructose at the isopolar level, but little or no sucrose (Klee and Giovannoni, 2011).
The accumulation of soluble sugars reflects the combined activity of the invertase acid
(Klann et al., 1996; Jin et al., 2009). CWIN and VIN are important regulators of plant
growth and development, and also play important roles in the plant's response to biotic
and abiotic stresses. is essential for carbon metabolism and development in fruit (Yelle
et al., 1991; Zanor et al., 2009). To ensure proper plant growth, the invertase activity
needs to be strictly controlled.
Due to the glycosylation process, the invertase acid is essentially stable
proteins and the regulation of the invertase acid may depend largely on post-transport
mechanisms (Jin et al., 2009; Zhang et al., 2015). CWIN and VIN can be directed at
the post-production level by compartment-specific inhibitory proteins (Rausch and
Greiner, 2004). CIFs, belonging to the family of proteins involved in the pectin methyl
esterase inhibitor (PMEI-RP) (Pfam 04043), are determined based on the activities of
invertase acids and regulate the efficiency of the invertase enzyme and signal flow
11


glucose down in vivo, thereby increasing sucrose content (Qin et al., 2016). The posttransition modulation of CWIN activity by AtCIF1 promotes seed germination by
refining the hydrolysis of sucrose (Su et al., 2016). In tomatoes, two CIFs, ie SlCIF1
and SlCIF2, were identified (Jin et al., 2009). Limiting CWIN or VIN activity by using
specific inhibitors may be a mechanism for achieving optimal cellular glucose levels
or the optimal hexose-sucrose ratio for proper growth (Li et al. , 2017). Increasing
CWIN activity by silencing SlCIF1 (Solyc12g099200) can increase tomato seed
weight and fruit hexose. However, direct evidence demonstrates that overexpression of
SlCIF1 in tomatoes has a similar inhibitory effect on the apoplasmic invertase activity

as observed in Arabidopsis (Jin et al., 2009).

Figure 2.3. CIF1 gene in Tomato

2.4. Biological techniques for gene editing technology
2.4.1. ZFNs and TALENs
ZFNs and TALEN are the first discovered gene editing techniques and have
brought a lot of value to scientific research.
Zinc finger nucleas (ZFNs) are artificial restriction enzymes created by fusing the zinc
finger DNA binding region with the DNA cleavage region (Porteus and Carroll, 2005).
Utilizing endogenous DNA repair machinery, ZFN can be designed to precisely alter
the genomes of higher organisms, allowing it to target almost any region of the
genome. It has improved gene targeting efficiency by introducing double-stranded
DNA breaks in endogenous HR-stimulating target genes (Porteus and Carroll, 2005;
Camenisch et al., 2008). Genetically engineered ZFN genes up to 7.7 kb in size were
efficiently introduced into the host genome of mammalian cells (Moehle et al., 2007),
and mutant mitochondrial DNA. Variables were also targeted to selectively and
degrade in cultured cells (Minczuk et al., 2008)) using this approach. The significant

12


enhancement in target gene substitution stimulated by ZFNs shows its potential for use
in silent cells such as hepatocytes. In fact, the in vivo genome technique has been
successfully used to correct factor IX deficiency (FIX) and prolonged bleeding in a
mouse model with hemophilia B using ZFNs (Li et al., 2011). rAAV vectors
associated with ZFN were also used to modulate bleeding in mice with adult
hemophilia (Anguela et al., 2013). The association of ZFN with the piggyBac vector
(PB) has been shown to modulate AAT deficiency in induced pluripotent stem cells
(iPS) (Yusa et al., 2011). ZFNs, however, have an inherent downside. Specifically,

assembling parallel zinc finger domains into a chain of high affinity is a challenge.
Additionally, laboratory procedures have proven time consuming for any laboratory
staff member who is not a ZFN specialist, resulting in a substantial effort required to
create a revision effective. The main advantage of the ZFN system is the use of
dimeified FokI, which can increase the specificity of DNA targeting and reduce offtarget effects.
TALEN consists of the FokI nuclease domain and the customizable DNA
binding domain derived from Xanthomonas transcription activator-like agents, a plant
pathogen (Miller et al., 2011). In the TALE structure, the DNA sequence recognition
region is characterized by a repeating unit of 33–35 conserved amino acids. Each
replication is almost identical except for the two highly modified amino acids at
positions 12 and 13, which are called repetitive modified amino acids (Boch et al.,
2009; Moscou & Bogdanove, 2009). A repeating unit recognizes a base pair in the
main groove of DNA (Deng et al., 2012; Mak, Bradley, Cernadas, Bogdanove, &
Stoddard, 2012). Like ZFNs, TALENs act as heterolecules because the Fok1 domain
requires isomerization in order to function. The whole-genome TALEN library,
targeting 18,740 human protein-coding genes (Kim, Kweon, et al., 2013) and 274
human miRNA-coding sequences (Kim, Wee, et al., 2013), has available. Compared
to ZFN, TALEN design has become much easier. The potential advantage of TALENs
is that the TALE array can be extended to the desired length, while the ZFN has
limitations on sequence length (Guilinger et al., 2014). This evidence suggests the use

13


of larger TALENs with less specificity. Another advantage of TALEN over ZFN is
that it has multiple structures (Reyon et al., 2012).
The size of TALENs is significantly larger than ZFNs which is the major
disadvantage. The size of cDNA encoding TALENs is 3 kb, whereas cDNA encoding
a ZFN size is only 1 kb. In practice, this makes it harder to deliver and express a pair
of TALENs when compared to ZFNs (Holkers et al., 2012). Designing of TALENs is

also very expensive, which cannot be afforded by small laboratories.
2.4.2. CRISPR/Cas9
Most recently, a new gene targeting tool has been developed in cluster-based
microbiological and mammal systems frequently interspersed with a short
palindromic-linked nuclease system (CRISPR). Nuclease binds to CRISPR as part of
adaptive immunity in bacteria and ancient bacteria (Deveau et al., 2010). Cas9
endonuclease, a component of the CRISPR / Cas system of Streptococcus pyogenes
type II, forms a complex with two short RNA molecules called CRISPR RNA
(crRNA) and metabolic crRNA (transcrRNA), which instructs the nuclease to cleave
DNA. not order on both strands. at a specific location (Gasiunas et al., 2012). The
Heteroduplex crRNA-transcrRNA can be replaced with a chimeric RNA (also known
as a pathogen RNA (gRNA)), which can then be programmed for specific targeted
sites (Jinek et al., 2012). The minimum restriction to the gRNA-Cas9 scheme is at
least 15-base pairing between the 5 'RNA designed and the target DNA without the
mismatch and NGG motif (called a contiguous motif protospacer or PAM) follows the
base mating region in the target DNA sequence (Kabin Xie, Yinong Yang., 2013). In
general, 15-22 nt at the 5 'end of the gRNA region is used to direct the Cas9 nuclease
to produce site-specific DSBs. The CRISPR / Cas system has been proven for human
genome editing (Cho et al., 2013; Cong et al., 2013; Jinek et al., 2013; Mali et al.,
2013b), mouse (Shen et al., 2013), zebrafish (Chang et al., 2013; Hwang et al., 2013),
yeast (Dicarlo et al., 2013), and bacteria (Jiang et al., 2013). Different from animal
cells, yeasts or bacteria, recombinant molecules (DNA, RNA or proteins) can be
transformed directly to plants.

14