INTRODUCTION1. 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 REVIEW8 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 METHODS2.1. 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 Technology2.2. 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 abilityStudied 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

x 100Transforming samples

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 DISCUSSION3.1. Evaluating studied maize cultivars’weevil hesistant abilityTo 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

Cultiv

arSL LC1 LC2 LC3 LVN99

S 1754,19 3515,75 2594,27 2128,14 6088,71

3.2. The characteristics of gene ZmDEF1 isolated from the studied maize cultivars3.2.1. The characteristics of gene ZmDEF1 (cDNA) isolated from the studied maize cultivars3.2.1.1. Cloning CDNA and gene ZmDEF1 sequence determinationBased 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 bpExon1- 64 bp Exon2- 179 bp

1 166 65 345

Figure 3.6. Maize ZmDEF1gene’s structure

3.2.3. The variety of maize ZmDEF1 gene’s encoding region sequencesTo 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 plants3.3.1. 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-SLHEP- ZmDEF1 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 root- developing 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

Experimenta

l

Samples

Transforme

d leaves

pieces of

leaves

Clusters

of buds

Alive

plants

Plant lines

germinate

d

Plant lines

grown in

the

greenhous

e

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 plants3.3.3.1. 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 plantsAnalysing 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 ZmDEF1protein 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 seedsSample Only

α -

amylase

DC Mixture of weevil α-amylase and

protein of T1 transgenic tobacco

seeds

T1-1 T1-3 T1-10 T1-17

α- amylase

activity (U/mg)

7,18

0,29

7,13

0,36

2,26

0,46

2,01

0,58

2,68

0,73

2,15

0,68

α- amylase

activity

performance

(%)

100 31,69 28,19 37,58 30,15

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 plants3.4.1. 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

Experimental groups

Number

of

embryos

transfor

med

Number

of

selected

callus

Number

of callus

regenerati

on

Number

of of

potting

plants

Number

of grouts

In winter

- spring

LC1 136 7 1 0 0

LVN99 147 8 1 0 0

(2015-2016)

In

Summer

(2016)

LC1 158 22 8 5 3

LVN99 167 23 7 4 2

DC0- LC1 30 3 0 0 0

DC0-

LVN9930 4 0 0 0

DC1- LC1 30 22 16 10 10

DC1-

LVN9930 20 13 10 10

(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 plants3.4.3.1. 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 plantsTransgenic 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

weevil

α -

amylase

Mixture of weevil α-amylase

and protein of T1 transgenic

LC1 seeds

Mixture of weevil α-

amylase and protein

of T1 transgenic

LVN99 seeds

s Non-

transg

enic

plants

Transgenic plants Non-

transge

nic

plants

Transgenic

plants

T1-C1 T1-

C3

T1-C5 T1-

L1

T1-

L3

α-amylase

activities

(U/mg)

7,98

0,67

5,1

2

0,59

1,89

0,62

2,23

0,37

2,11

0,47

5,87

0,65

2,6

7

0,29

2,6

3

0,43

Performan

ce α -

amylase

(%)

100 36,91 43,55 41,21 100 45,4

8

44,8

0

α- amylase

activity

performan

ce (%)

0 63,09 56,45 58,79 0 54,5

2

55,2

0

CONCLUSIONS AND RECOMMEDATION1. Conclusion1.1. 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.