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INTRODUCTION
1. Statement of the Problem
Maize (Zea mays L.) is one of the major cereal crops with high yield and
economic value, providing 30% of the world’s population. Nowadays, maize
ranks third in planted area, second in yield and first in productivity worldwide.
Maize has been used to produce approximately 650 types of goods in the food,
medical and light industries. Maize contributes to the steady production of
cereals in the world and has an important role in economy and international trade.
In Vietnam, the developing of maize over recent years has gained many
achievement, increasing in size, productivity and yield; despite that, it has faced
some challenges, one of which is weevils. Though many new hybrid maize
cultivar with high productivity has been put into practice, there has also been a
number of limitations regarding storage ability such as vulnerability to mould,
weevils, etc... while plenty of native maize cultivars can be stored longer after
being harvested despite low productivity.
Maize weevils (Sitophilus zeamais Motsch.) eat most types of cereals,
legumes, oil seeds, and other plant products, though they prefer corn. The most
suitable food for them is maize kernels. Weevils heavily affect the yield and
productivity of maize, reducing 20% of medium harvested crop, in some cases,
their damage can reach 90% in six months. Preventing maize weevils infestation
is an urgent issue in order to protect the world’s food safety nowadays. Current
preserving method such as drying, mixing with inert dust, or stirring are
time-consuming and inefficient, while chemical treatment may cause risks of
environmental pollution and poisoning for humans and animals.
Plant defensins which have small globular spatial structure containing
about 45-54 cysteine-rich amino acid widely distribute among vegetation.
Defensines are multifuncional proteins and able to inhibit decoding, protease
activities and especially insect’s α-amylase activities as well. They can weaken
microorganisms, increase resistance to zinc and change oxygenation conditions.
It was reported in many studies that defensinces isolated from chickpea and
cowpea have ability to inhabit the α-amylase activities of weevil larvae in those
peas. These defensines’ inhibition to vertebrates, however, is not very effective.
Therefore, many scientists have done researches on improving fabaceae’s weevil
resistance using protein defencines (PD). Up to now, neverthless, there have no
studies into applying PD to other agricultural products damaged by weevil such
as maize.
Recently, genetic modification (GM) has been an useful tool in
conducting researches on vegetation as well as breed improvement. Transgenic
techniques have made a great progess since the first success in transfering
foreign genes into plants using A. Tumefaciens. Transgenic plants have special
attributes such as improving nutrition values, increasing yield, strengthening
resistance and many other preeminences. Hence, applying transgenic engineering
to create high weevil resistance maize cultivars is a pratical issue in preservation.
Thus, the goal of this thesis is to study the attributes and gene expressions
relating to weevil resistance isolated from maize.
2. Objectives
To determine the attributes of ZmDEF1 isolated from several maize cultivars
with different resistant abilities to weevils. To express protein recombination
ZmDEF1 (rZmDEF1) in transgenic plants.
3. Contents
(i) Evaluate resistance to weevils of studied maize cultivars in artificial
contagious conditions.
(ii) Analyse ZmDEF1 gene’s attributes of maiz cultivars with different
resistances to weevils.
(iii) Create transgenic vector containing ZmDEF1 and evaluate their activities in
tobacco T0 and T1 transgenic generations, analyse rZmDEF1’s α-amylase
inhibitory ability in maize weevil larvae.
(iv) Create ZmDEF1 transgenic maize cultivar from LC1 and LVN99. Analyse
ZmDEF1 gene’s expression in T1 maize transgenic generation and evaluate
rZmDEF1’s α-amylase inhibitory ability in maize weevil larvae.
4. Contributions of the thesis
This thesis is a systemetic study which starts from evaluating maize
cultivars’ weevil resistance to gene isolation, cloning, sequencing, creating
transgenic vectors and analysing ZmDEF1 gene’s expressions in transgenic
tobacco and maize.
Particularly:
1. After the researcher evaluated maize cultivars’ weevil resistance, SL
cultivar was found to have the best resistance to weevils while LC1 and LVN99
cultivars have low resistant ability.
2. ZmDEF1 gene (DNA)isolated from studied maize cultivars has 345 bp,
it is made of an intron with 102 bp between two exons; ZmDEF1 gene (cDNA)
has 243 bp codifying 80 amino acids.
3. ZmDEF1 gene was successfully espressed in both transgenic tobacco
and maize. With the atom weight of about 10 kDa, rZmDEF1 protein extracted
from transgenic tobacco and maize plants expresses maize weevil larvae’s
α-amylase inhibition
5. Scientific and pratical significance of the thesis
The findings of the thesis have both scientific and pratical values in
studying on improving weevil resistance using transgenic engineering.
Scientific significance
What was found in the thesis made a contribution to clarify the structure
of ZmDEF1 gene isolated from local and hybrid LVN99 maize cultivars,
determine ZmDEF1 gene’s structure in maize through comparing nucleotide
sequences isolated from cDNA and DNA.
This thesis is also a scientific foundation for transgenic application to
increase weevil resistance in maize.
The articles which were published in national and international science
and teachnology magazines as well as gene sequences registered in gene bank are
useful references for both studying and teaching.
Practical significance
The products obtained from the study have laid a literature foundation for
researches on increasing weevil resistance by enforcing ZmDEF1 gene’s
expression through transgenic methods and creating new high weevil resistance
maize cultivars in the future.
6. Structure of the thesis
The thesis is divided into three main parts: Introduction (4 pages);
Development (102 pages, consists of 3 chapters: Chapter 1- Literature Review,
Chapter 2 – Materials and Methods, Chapter 3- Findings and Discussion);
Conclusion and Recommendations (1 page). There are also lists of published
scientific works relating to the topics (2 pages) and references (17 pages) in the
appendix. The thesis contains 30 tables and 33 images.
Chapter 1. LITERATURE REVIEW
8 referential documents in Vietnamese as well as other 135 ones in
English have helped the researcher have an overview about relating matters as
folowing: (1) maize weevils (Sitophilus zeamais Motsch); (2) the attributes/
characters of plant defensines; and (3) Transgenic engineering in maize plants.
Maize weevil is a popular destructive species and protopathic pest. It is
distributed globally, mainly in warm areas and especially in Asia, Mediterranean
region and North America. In Vietnam, maize weevil is found nationwide and in
every maize storage. On evarage, weevils makes a 20% reduction in weight of
the total maize havested crop, less than 20% and even 90% in good and low
quality storages respectively. The harder and thicker the shell is as well as the
phenolcarboxylic acid richer, the higher tripsin inhibitor and higher fibre the
maize contains, the better its hesitance to weevils is.
Defensine is the peptide cation which consists of 45-55 amino
acids including eight conserved cysteine residues. The three-dimensional
structure of defensin is composed of 3 β-sheets and one α-helix. Loop 3, which is
in the middle of the second and third β-sheets, plays an important role in the
inhibition of weevil α-amylase activities. The interaction between loop 3 (VrD1)
and the active site of weevil α-amylase prevents starch from entering that site,
which weaken the weevils to death. (Lin et al., 2007). However, this inhibition
has little expression among animals’ due to longer mammals’ 3 loops in active
site of α-amylase, which prevents defensine loop 3 (VuD1) from connecting to
active site of α-amylase.
Embryos are proved to be the best for maize transgene using A.
Tumefaciens. Both crop yield and nutritious value have been improved a lot
thanks to the introduction of transgenic maize cultivars into production. So far, a
variety of protein-coding genes such as Cry of Bacillus thuringiensis have been
successfully tranfered into maize plants (Liliana et al., 2013). There are also
cultivars with high resistance to drought (Qiudeng et al., 2014), productive
transgenic cultivars with either heavier wieght or greater numbers of seeds
(Zhang et al., 2016), and multi-vitamin transgenic cultivars (Shaista et al., 2009).
However, despite those archievements, the study of transgenic maize cultivars
with high resistance to weevils has been paid little attention.
Chapter 2 – MATERIALS AND METHODS
2.1. Materials
4 local and 1 hybrid LVN99 maize cultivars provided by Lao Cai Plant
Seeding Center and Son La Plant Seeding Center;
Nicotinana tabacum C9-1 provided by Vietnam Tobacco Technical and
Economic Institute;
Escherichia coli DH5α, A. tumefaciens CV58; gene cloning vector pBT,
p201- SLHEP- HA, pBetaPhaso-dest provided by Institute of Biotechnology,
Vietnam Academy of Science and Technology
2.2. Chemicals, Equipment and Places of Study
Chemicals and KITs are purchased from prestigious manufacturers such
as Bio-Neer, Fermentas, Invitrogen, ect.
Experiments to test weevil hesistant ability and biological functions of
tranfered genes were carried out at the Laboratory – Faculty of Biology – TayBac
University. Other experiments on isolating genes, creating vectors, transforming genen
into tobacco plants and transgenic analysing, etc. were carried out at Department of
Applying ADN Technology, Department of Plant Cell Technology and the Key
Laboratory of Gene Technology, Institute of Biotechnology, Vietnam Academy of
Science and Technology. The experiments on gene transformation into maize plants
were carried out at the Department of Cells and Modern Biology, Faculty of Biology,
Thai Nguyen University of Education, Thai Nguyen University.
2.3. Methods of Study
Researching methods were divided into 5 main groups: (1) evaluating studied
maize cultivars’weevil hesistant ability, (2) cloning and determining gene
sequence, (3) creating plant transgenic vector, (4) creating transgenic plants and
(5) analysing transgenic plants.
2.3.1. Evaluating studied maize cultivars’weevil hesistant ability
Studied corn grains (without diseases) were dried to 13% of moisture then
divided into 2 samples: the experimental and the control. 50 gram of
experimental grains were put into a lidded jar together with 15 pairs of maize
weevils while other 50 gram of control grains were put into a lidded jar without
weevils. After 15 days, 30 days and 45 days, such criterials were determined
among experimental samples as: weight loss, incidence rate, the proportion of
generated cornmeal and coefficient of weevil population increase (r).
2.3.2. Cloning and determining gene sequence
ZmDEF1 gene was isolated by PCR with specifically designed primers
ZmDEF1F_SalI/ZmDEF1R_HindIII and total mold DNA extracted from the
leaves; RNA extracted from the leaves soaked with ABA through cDNA
synthesis.
Cloning and sequencing through the steps: (i) PCR product purification,
(ii) Pairing gene segments to cloned vector pBT, (iii) Transformating
recombinant plasmid to variable cell E. coli DH5α, (iv) Selecting colony by PCR,
(v) Plasmid extraction and test cutting with BamHI, (vi) Nucleotide sequence
identification and analysis.
Total RNA was isolated from maize by the use of TRIzol reagent (Life
Technologies). cDNA was synthesized by using Maxima Fiencerst Strand cDNA
Synthesis Kit (ThermoScientific). The ZmDEF1 gene was cloned from cDNA by
using the ZmDEF1-F and ZmDEF1-R primers. Findings on the gene sequence,
amino acids were processed by such softwares as BioEdit, DNA Star.
2.3.3. Creating plant transgenic vector ZmDEF1
Vector containing transgene ZmDEF1 was developed in two basic steps: (1)
Creating structure containing transgene pDON201-SLHEP-ZmDEF1, (2)
Attaching the gene structure ZmDEF1 transgene into the transgenic vector
pBetaPhaso-ZmDEF1.
2.3.4. Creating transgenic plants by A.tumefaciens
pBetaPhaso-ZmDEF1 was transformed into A. tumefaciens cells with
electric pulses CV58, selected by colony PCR and cloning vectors to create
transgenic strains carrying the gene transfer vector containing structural
transformation ZmDEF1.
Transformation of the transgenic structure containing ZmDEF1 in tobacco
plant C9-1 by A. tumefaciens was performed according to Topping’s protocol
(1998
Transfer vector containing the transgene structure ZmDEF1 into maize
embryos LC1 and LVN99 by bacteria A. tumefaciens. The process to create
transgenic soybean plants is based on research done by Frame et al, 2002 and
regenerating process to create transgenic soybean plants by Tran Thi Luong et al,
2014.
Transgenic productivity (%) =
Total plants containing transgenes
Transforming samples
x 100
2.3.5. Transgenic plant analysis
Check the presence of the transgene by PCR with specific primers
ZmDEF1-SalI/ZmDEF1R-HindIII, Southern hybridization techniques. Check the
presence of the gene transcription by RT-PCR ZmDEF1. Assess the level of
expression of the transgene transcription by Real-time RT-PCR according to
method of R = 2-ΔΔCt by Livak, 2001. Check the presence of rZmDEF1 protein
by Western hybridization. Evaluate biological functional expression of the
transgene in its ability to inhibit weevil larvae’s α-amylase activity of protein
rZmDEF1.
Chapter 3 – FINDINGS AND DISCUSSION
3.1. Evaluating studied maize cultivars’weevil hesistant ability
To determine the studied maize’s resistance to weevil (Sitophilus zeamais
Motsch.), at the same time, to have background for the choice of the best weevil
resistant maize, and to serve gene isolation in order to provide raw materials for
the development of gene transfer vectors, we perform reviews of resistant
varieties of corn weevil research in artifical infectioious condition of 4 local
varieties : LC1 LC2 LC3, SL, and maize seed LVN99, such criterials were
determined among experimental samples as: weight loss, incidence rate, the
proportion of generated cornmeal and coefficient of weevil population increase
(r).
Analysis of data presented in the tables 3.1, 3.2, 3.3, 3.4, 3.5 of the thesis and the
results showed that SL cultivar is the best weevil resistant, LVN99 and LC1
cultivars have low weevil resistance.
Table 3.5. Weevil relative sensitivity indicator (S) of the studied maize cultivars
Cultivar
SL
S
1754,19
LC1
3515,75
LC2
LC3
2594,27
2128,14
LVN99
6088,71
3.2. The characteristics of gene ZmDEF1 isolated from the studied maize
cultivars
3.2.1. The characteristics of gene ZmDEF1 (cDNA) isolated from the studied
maize cultivars
3.2.1.1. Cloning CDNA and gene ZmDEF1 sequence determination
Based on the maize gene ZmDEF1 sequence published in the international gene
bank
carrying
Code
JF797205,
specific
primers
ZmDEF1F_SalI
/
ZmDEF1R-HindIII containing restricted enzyme cutting point SalI and HindIII
for vector design, PCR products magnified from cDNA gene ZmDEF1 were
tested on agarose gel DNA received get a DNA tape about 0, 25kb in size
consistent with the predicted size when designing PCR primer pairs (Figure
3.2-A). ZmDEF1 gene cloned with these steps: pBT attached to the vector; DH5α
transformed into E. coli by heat shock; colony PCR containing target gene to
select line recombinant using primers M13_F / M13_R (Figure 3.2-C). Before
sequencing, gene recombinant plasmid ZmDEF1 was checked by BamHI cut to
5/5 positive samples. Sequencing on the machine is done automatically repeated
3 times / sample. The analytical results show that the gene encoding segments in
the studied ZmDEF1 of size 243 bp, encoding 80 amino acids, with the similarity
of genome sequences is 98.8% to 99, 6%. So it can be claimed that gene
ZmDEF1 (cDNA) has been successfully isolated and cloned from studied maize
cutivars.
3.2.1.2. Analysing the nucleotide sequence in encoding region of gen ZmDEF1
Results conducted after comparing nucleotide sequences of gene
ZmDEF1 isolated from local maize cultivars and hybrid cultivar LVN99 with
sequence Code JF797205 on international gene bank show that among gene
ZmDEF1sequences there are 5 nucleotide positions different from the nucleotide
position No. 43, 53, 101, 161 and 195.
Amino acid sequences deduced from studied maize
ZmDEF1 gene’s
encoding region and JF797205’s both consisit of 80 amino acids as in Figure
3.4. Figure 3.4 shows that, There are 2 wrong amino acid positions: position 3,
23 in the functioning region of the studied maize. Particularly, amino acid at the
3rd position of local maize cultivars SL, LC1, LC2 , LC3 and JF797205 are
cysteine (C) to form the disulfide bridge with the 49th Cys, while in hybrid maize
LVN99, the 3rd position is tyrorine (Y). So in the ZmDEF1 protein structure of
hybrid maize LVN99 will only 3 disulfide bridges.
Figure 3.4. Amino acid sequences deduced from studied maize ZmDEF1
and JF797205 on international gene bank.
3.2.2. Characteristics of ZmDEF1 gen isolated from DNA
The results after comparing ZmDEF1gene sequences isolated from total DNA
with the sequences isolated from cDNA showed that ZmDEF1 in genome is 345
bp in length and consists of two exons and intron 1 interspersed with 102 bp
(Figure 3.6).
Intron – 102 bp
Exon1- 64 bp
1
65
Exon2- 179 bp
166
345
Figure 3.6. Maize ZmDEF1gene’s structure
3.2.3. The variety of maize ZmDEF1 gene’s encoding region sequences
To determine the level of diversity in the nucleotide sequence of the ZmDEF1
gene, we perform comparisons in ZmDEF1 gene encoding region sequence of 14
published ones in the international gene bank. Results showed that 14 sequences
of ZmDEF1 gene encoding region divided into two large groups with the genetic
distance of 2.8%.
3.3. ZmDEF1 gene expression in T1 transgenic tobacco plants
3.3.1. Constructing the structure containing transgene ZmDEF1
Nucleotide sequence of the cultivar SL’s gene ZmDEF1 was used to
design plant transgenic vector through A. tumefaciens. Transgenic vector carrying
transgene ZmDEF1 was developed in two steps: (1) Construct the structure
carrying the transgene pDON201-SLHEP-ZmDEF1, pDON201-SLHEPZmDEF1 containing crossing-over sequence attL; (2) Attach the gene structure
containing ZmDEF1 transgene to the transgenic vector ZmDEF1 pBetaPhaso-LR
completely with LR reaction with pBetaPhaso-dest which has attR exchanging
sequences with attL to form binary vector pBetaPhaso- ZmDEF1 is cloned into
variable cell E. coli DH5α. Colony-PCR reactions were performed using primers
ZmDEF1F_SalI/ZmDEF1R_HindIII. The positive colonies with colony-PCR
reactions were used to isolate recombinant plasmid pBetaPhaso-ZmDEF1 and are
cut by Hind III restricted enzymes to check for the presence of recombinant
vector ZmDEF1 in (Figure 3. 8-A). After cutting plasmid and cutting by
restricted enzymes Hind III DNA we obtained 3 Tape size about 1.85 kb, 2.4 kb
and 11.5 kb. The size is completely consistent with the theoretical calculations.
Figure 3.8. Image electrophoresis products by recombinant plasmid cut Hind III (A) and
electrophoresis product image colony-PCR using primers ZmDEF1F_SalI / colony A.
tumefaciens ZmDEF1R_HindIII CV58 (B)
After being checked the recombinant vector bearing pBetaPhaso-ZmDEF1
structure was transformed into bacterial cell CV58 A.tumefaciens by electrical
impulses engineering. Colony-PCR reactions using specific primers
ZmDEF1F_SalI/ZmDEF1R_HindIII were performed (Figure 3.8-B) shows that
all of the 3 CV58 A. tumefaciens bacteria lines contain vector bearing
pBetaPhaso-ZmDEF1 structure and they are used to transfer genes into plant
cells through infection.
3.3.2. Transform pBetaPhaso-ZmDEF1 structure into tobacco plants by A..
tumefaciens
Transform the structure pBetaPhaso-ZmDEF1 into N. tabacum C9-1
tobacco plant thanks to A.tumefaciens twice through stages: synchronic
transplatation, multi-bud regeneration, select in such stages as bud stretching,
rooting , taking out of land and into the greenhouse (Figure 3.10).
Figure 3.10: Regeneration and pBetaPhaso-ZmDEF1 structure
transformation into C9-1 tobacco plant
(A: transplanted pieces of the leaves after bacteria washing in selective
environment; B: The clusters of buds are formed after two weeks of
transplantation in selective environment; C: The green buds are separated on
in selective environment; D: transgene sprouts after 3 weeks in rootdeveloping environment; E: root morphology of transgene tobacco plant in
selective environment; F: transgene tobacco plant flowering
After 2 times transforming pBetaPhaso- ZmDEF1 structure into
pieces of tobacco leaf (30 pieces each time), the results are presented in Table
3.9 with 18 lines of transgenic plants to the greenhouse.)
Table 3.9. pBetaPhaso-ZmDEF1 structure transformation into C9-1
tobacco leaves
Experimental
Transformed
pieces of
Clusters
Alive
Plant lines
Plant lines
Samples
leaves
leaves
of buds
plants
germinated
grown in
the
greenhouse
Expriments
2 x 30 = 60
32
68
98
36
18
DC0
30
0
-
-
-
-
DC1
30
30
72
102
10
10
(DC0: non-transgenic tobacco grown in regeneration environment
supplemented with antibiotics; DC1: non-transgenic tobacco are grown
regeneration environment without antibiotics)
3.3.3. Analysis of transgenic tobacco plants
3.3.3.1. Determining the presence of transgene ZmDEF1 in T0 transgenic tobacoo
plants
To check the presence of transgene ZmDEF1 in T0 generation 18
transgenic tobacco lines, we extract DNA of leaves of 18 transgenic tobacco lines
grown in a greenhouse to perform PCR with specific primers ZmDEF1F_SalI /
ZmDEF1R_HindIII. Results of agarose gel electrophoresis of PCR products are
displayed in Figure 3.11 with 13/18 lines positive with PCR.Transgene
performance at this stage is 13/60 = 21.67%.
Figure 3.11: Results of agarose gel electrophoresis of PCR products
(M: 1kb DNA standard scale; 1-18: transgenic tobacco lines; (-):
non-transgenic control plants; (+): PCR from transgenic structure
pBetaPhaso-ZmDEF1)
3.3.3.2. Analysis of ZmDEF1 gene expression in T1 transgenic tobacco plants
Analysing ZmDEF1 expression in T1 transgenic tobacco plants at the
level of transcription by RT-PCR reaction. The seeds of the tree lines positive
with PCR reaction were used to perform RT-PCR reaction (Figure 3.12).
(M: 1kb DNA standard scale; 1-18: transgenic tobacco lines; (-):
non-transgenic control plants; (+): PCR from transgenic vector
pBetaPhaso-ZmDEF1)
Figure 3.1. Gene ZmDEF1 expression levels from
T1 transgenic tobacco plant lines
(T1-1, T1-3, T1-10, T1-17: T1 transgenic tobacco plant lines; vertical axis:
standard error)
The transcript expression levels of the transgene in the 4 positive T1
transgenic lines were quantified. Tobacco seeds from lines T1-1, T1-3, T1-10,
T1-17, and nontransgenic (negative control) were used to perform realtime
RT-PCR with qDEF1-F/qDEF1-R primers. (The cycle threshold (Ct) of the
negative control was after the 40th cycle, which means that the tested sample did
not carry ZmDEF1. Results of analysis showed that the rate of gene ZmDEF1
expression in T1 transgenic tobacco plant lines T1-1, T1-10, T1-19 are
respectively 1.17 times, 1.22 times and 1.44 times higher than T1-3 (Figure 3.14)
Analysis of recombinant protein ZmDEF1 by Western blot
Figure 3.15. Western blot for recombinant ZmDEF1 protein in T1
generation transgenic tobacco plants.
(M: Standard protein scale (10–250 kDa); 1, 3, 10, 17: recombinant ZmDEF1
protein of T1 transgenic tobacco plant lines (T1-1, T1-3, T1-10, T1-17); (+):
protein, ~35 kDa, with c-myc tag; (-): protein of nontransgenic plant.)
Figure 3.15 shows that all four transgenic lines expressed recombinant
defensin 1 protein. Thus, it could be concluded that the four transgenic tobacco
lines obtained successfully expressed rZmDEF1.
3.2.3. 4. Analyze the biological function of gene ZmDEF1 in T1 transgenic tobacco
seeds
Check rZmDEF1 protein biological function by determining its ability to
inhibit the weevil α -amylase activity.
Table 3.1. Analysis on the ability to inhibit maize weevil α-amylase of
recombinant protein ZmDEF1 from T1 transgenic tobacco seeds
Sample
Only
DC
Mixture of weevil α-amylase and
α
protein of T1 transgenic tobacco
-amylase
seeds
T1-1
α-
amylase 7,18
activity (U/mg) 0,29
α-
amylase
 7,13 
0,36
100
2,26 
0,46
31,69
T1-3
2,01
T1-10
T1-17
 2,68
 2,15
0,73
0,68
0,58
28,19
37,58

30,15
activity
performance
(%)
DC: Mixture of weevil α-amylase and protein of non transgenic tobacco seeds
Table 3.11 shows, with α-amylase activity performance of α-amylase
from larvae and protein of non-transgenic tobacco plants, the α-amylase activity
of the samples containing the mixture of protein the gene transfer decreases
tobacco plant line, only 28.19 percent to 37.58%.
3.4. Gene ZmDEF1 expression in transgenic maize plants
3.4.1. Gene gus transformation into cultivar LVN99 embryos
The influences of A. tumefaciens density, concentration of
acetosyringone (AS), the time of infection, the age of the embryo transgenic
maize Gus embryo and selection threshold of embryos carrying the transgene in
maize LVN99 kanamycin were identified, to set the foundation for successful
gene transformation into maize ZmDEF1 embryos. Embryos 10-12 days old age
(the size 0,9- 1,3 mm) is the optimal age of embryos capable of receiving the
gene and rebirth.The results are presented in table 3.12, 3.13, 3.14 in the thesis
shows, the process of embryo transfer through the small GUS A. tumefaciens
strain of maize by C58 has been developed and optimized with A. tumefaciens
bacterial density appropriate at OD660nm = 0.8 and values infections optimal
time of 30 minutes, an additional aS 150 Selective antibiotic kanamycin
threshold for transgenic embryo is 50 mg / l.corresponding to
3.4.2. Transform structure containing ZmDEF1 into maize embryos by A..
tumefaciens
LVN99 hybrid maize and maize LC1 local material to get the gene transfer of
ZmDEF1. The process variable load, rebirth created maize gene transfer are
illustrated in Figure 3.17.
Figure 3.17. The image regeneration of transgenic corn from embryos
green corn seed ZmDEF1 LVN99 summer - 2016 collection
(A1: Embryos after bacterial infection are pets on board environmental
implications; A2: the Embryos feed on on antibiotic selection environment
kanamycine 50 mg/l, select embryos carrying genes that switch; A3: the
Embryos feed on antibiotic selection environment kanamycine 25 mg/l, selective
embryo carry the gene transfer 2 times; A4: the Embryos feed on scar tissue
recovery environment;A5: Embryonic pets on the environment regeneration
buds; A6: Embryonic pets on root environments; Trees: Trees are grown
outdoors).
After twice transformed immature embryos of maize used in the winter spring (November 2015 to 2 in 2016) and summer - collection (April 5 August
2016) we obtained results of transformation and regeneration two GM crops of
maize and LVN99 LC1 at 3:15 with 5 tables transgenic plants from seed LC1,
LVN99 4 seed crops from the potting soil.
Table 3.2. The result tree regeneration gene transfer of ZmDEF1 maize LC1
and LVN99
Number
Number
Number
Number
of
of callus
of of
Number
selected
regenerati
potting
of grouts
callus
on
plants
of
Experimental groups
embryos
transfor
med
In winter
- spring
LC1
136
7
1
0
0
LVN99
147
8
1
0
0
LC1
158
22
8
5
3
LVN99
167
23
7
4
2
DC0- LC1
30
3
0
0
0
30
4
0
0
0
30
22
16
10
10
30
20
13
10
10
(2015-2016)
In
DC0-
Summer
LVN99
(2016)
DC1- LC1
DC1LVN99
(DC0- LC1: LC1 corn maize embryos not implanted transgenic
regenerative environment supplemented with antibiotics; DC0- LVN99:
embryonic non-transgenic corn seeds implanted LVN99 on environmental
regeneration supplemented with antibiotics; DC1- LC1: embryonic
non-transgenic corn seeds implanted LC1 environment no additional
regeneration antibiotics; DC1- LVN99: embryonic non-transgenic corn seeds
implanted LVN99 environment no additional regeneration antibiotics)
3.4.3. Determining the presence of transgene ZmDEF1 in T0 transgenic maize
plants
3.4.3.1. Determining the presence of transgene ZmDEF1 in T0 transgenic maize
plants by PCR reactions
Total DNA from the leaves of transgenic maize 9 of two varieties LC1 (5
kilometers) and LVN99 (4 plants) after being purified, perform PCR Figure
3:18. Results showed that all 5 plants are transgenic varieties LC1 for DNA results 2
tape size 0.25 kb and 0.35 kb corresponding to the estimated size for intrinsic gene
and gene ZmDEF1 exotic ZmDEF1 . Performance of similar transgenic LC1 to the
evaluation period is 5/158 = 3.16%. The transgenic plant varieties positive LVN99 3
trees.Performance of transgenic seed stage LVN99 determine the presence of the
transgene by PCR is 3/167 = 1.79%.
Figure 3.18. Results PCR products electrophoresis to determine the presence of
the transgene ZmDEF1 in transgenic maize
(C1 C5: the transgenic maize varieties T0 LC1the system; L1- L4: The
transgenic maize varieties will LVN99 generation T0; M: 1kb DNA ladder
standards; (+) gene transfer vector PCR products ZmDEF1 ; (-): PCR products
from distilled water; WT: PCR products from non-transgenic maize DNA)
3.4.3.2. Determining the presence of transgene ZmDEF1 in T0 transgenic maize
plants by Southern blot
The transgenic maize LC1 and LVN99 positive with PCR was test to find out
the presence of the transgene and the number of copies in the genome by Southern
hybridization, the results shown in Figure 3:19.
Figure 3.19. Southern hybridization for T1 transgenic ZmDEF1 maize
plants
(M: Marker 1kb, (+) vector pBetaPhaso-ZmDEF1 carrying gene nptII; (-):
non-transgenic maize LVN99; C1 C5: The T0 transgenic maize LC1; L1, L3,
L4: the T0 transgenic maize LVN99)
The results showed that 5 transgenic plants LC1 were positive with
Southern. Transgenic performance up to the time of Southern analysis was
5/158 = 3.16%. LVN99 has 3 plants positive with PCR (T0-L1, L3 T0-, T0-L4)
and all 3 transgenic plants were positive with Southern, each tree has one
copy. Transgenic Performance of LVN99 is 3/167 = 1.79%.
3.4.4. Analysis of protein ZmDEF1 expression in T1 transgenic maize plants
Transgenic LC1 has 3 scobicular plants (T1-C1, C3-T1, T1-C5). Transgenic
LVN99 has 2 scobicular plants T1 and T1-L3-L1 and the second one doesn’t
contain transgene. Protein of transgenic maize seeds is extracted to perform
Western blot to check gene activities at the level of transgene transcription,
(figure 3:20.)
Figure 3:20. Western blot for T1 transgenic ZmDEF1 maize plants
(M: 10- 250 kDa protein standard scale; C1, C3, C5: The T1 transgenic maize
cultivar LC1, L1, L3: The T1 transgenic maize cultivar LVN99; (+): protein
containing c myc tail about 35 kDa in size; (-): protein of non-transgenic maize
LVN99)
Transgenic performance to transcriptional stage of LC1 is 3/158 = 1.89% and
LVN99 is 2/167 = 1.19%.
3.4.5. Analyze the biological function of recombinant gene ZmDEF1 in T1
generation
Evaluate T1 transgene maize seeds’ ability to inhibit α-amylase activity of maize
weevil larvae (table 3.16 )
Compared to non-transgenic cultivar LC1, weevil larvae α-amylase inhibitory
performance of proteins rZmDEF1 in T1-C1, T1-C3, and T1-C5 was 63.09%,
56.45% and 58.79% respectively. For cultivar LVN99 weevil larvae α-amylase
inhibitory performance of proteins rZmDEF1 in T1-L1and T1-L3 was 54.52%
and 55.20%.
Table 3.2. Analysis on the ability to inhibit maize weevil α-amylase of
recombinant protein ZmDEF1 from T1 transgenic maize seeds
Samples
Only
Mixture of weevil α-amylase
Mixture of weevil
weevil
and protein of T1 transgenic
α-amylase and protein
α
LC1 seeds
of T1 transgenic
-amylas
es
LVN99 seeds
Non-t
Transgenic plants
ransg
enic
T1-C1
plants
T1-C T1-C5
Non-tra
Transgenic
nsgenic
plants
plants
3
T1-L
T1-L
1
3
α-amylase
7,98 
5,12
1,89
2,23
2,11
5,87 
2,67
2,63
activities
0,67
0,59



0,65


0,62
0,37
0,47
0,29
0,43
36,91
43,55 41,21
45,4
44,8
8
0
(U/mg)
Performan
ce
α
amylase
(%)
100
-
100
α- amylase
activity
0
63,09
56,45 58,79
0
54,5
55,2
2
0
performan
ce (%)
CONCLUSIONS AND RECOMMEDATION
1. Conclusion
1.1. Local maize cultivar SL is best resistant to weevils , sensitive indicator is
1754.19; maize cultivar LVN99, LC1 are pooy - resistant to weevils, sensitive
indicators are 6088.71 and 3515.75 respectively.
1.2. Gene ZmDEF1 of the studied maize cultivars has been successfully cloned
and nucleotide sequence has been identified. Gene ZmDEF1 has two exons with
243 bp in size, coding for 80 amino acids and one intron containing102 bp
1.3. pBetaPhaso-ZmDEF1 structure is transformed into tobacco plants N.
tabacum C9-1 thanks to A. tumefaciens CV58. Protein rZmDEF1 expression was
in 4 transgenic lines (T1-1, T1-3, T1-11, T1-17) and has the ability to inhibit the
activity of α-amylase maize weevil larvae.
1.4. pBetaPhaso-ZmDEF1 structure was successfully transformed and created
transgenic maize T1 generation from local maize cultivars LC1 and LVN99 with
transgenic performance respectively 1.89% and 1.19%.
1.5. Protein rZmDEF1 nearly 10 kDa in size was expressed from the LC1’s 3
lines (T1-C1, T1-C3, T1-C5) and 2 lines from the cultivar LVN99 (T1-L1, T1-L3). Protein
rZmDEF1 expression inhibits α-amylase activity of the maize weevil
larvae. Compared to non-transgenic protein, protein in the transgenic lines
rZmDEF1 performing α-amylase inhibitor from 54.52% to over 63.09%.
2. Recommendation
Further analysis of the transgenic maize plants of culvivars LC1 and LVN99
through the T2, T3, ... to select, create stable tree line about the ability to inhibit
activity of α-amylase of maize weevil, fostering weevil resistant maize cultivars.