CREATING DROUGHT TOLERANCE LINES AND ISOLATING CYSTATIN GENES RELATED TO DROUGHT TOLERANCE IN PEANUT (Arachis hypogaea L.)

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MINISTRY OF EDUCATION AND TRAINING
THÁI NGUYÊN UNIVERSITY

VŨ THỊ THU THỦY

CREATING DROUGHT TOLERANCE LINES
AND ISOLATING CYSTATIN GENES RELATED TO DROUGHT
TOLERANCE IN PEANUT (Arachis hypogaea L.)

Major: Genetics
Code: 62.42.70.01

PHD THESIS ABTRACT

Thai Nguyen - 2011

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INTRODUCTION
1. Reasons for choosing the topic

Groundnut is a valuable crop in many aspects and many countries around the world
wishing to expand and develop groundnut production. Groundnut plant belongs to the bean
group with low drought tolerance. Compared to many other crops, groundnut specially
needs water as its roots do not have absorbing hair, groundnut fruit is shaped underground.
Statistical results show that groundnut production accounts for 40% of total cultivated land
area of short-term industrial crops, of which 2/3 depends on the rain. For many localities,

groundnut is the main crop, investment in the development of groundnut production
however has not yet been commensurate with its inherent potential.
There are many methods of improving plant varieties of which tissue culture of plant
cells is an effective technique, allowing applications and improvement of many features of
the plant. During the culture, the cells can be genetically modified because of the influence
of the environment. If mutation generated agents are combined, the mutation generated
frequencies will be significantly increased. This is significant in creating the source material
for breeding. Effectiveness of application of plant cells technologies to improve the
tolerance to externally adverse conditions is continuously confirmed. In Vietnam, the birth
of two rice varieties DR1 and DR2 which resist the drought and the cold primarily
demonstrates the possibility. Following is the research to improve drought tolerance and
salt-resistance of sugarcane by Yadav et al. (2006) and of wheat by Abdelsamad et al.
(2007), etc.
Drought tolerance of the plant is characteristics specified by multiple genes therefore
the search and analysis of genes related to drought tolerance are studied by many scientists.
Several genes related to drought tolerance of plants has been isolated and published such as
LEA genes in soybean and green beans, P5CS gene in soybean, cystatin genes in green bean
plants, DREB genes in Arabidopsis, etc. Cystatin gene (Cys) of vegetation was first
published on rice by Abe et al. (1987), so far Cys gene has been isolated in many species of
higher plants in both monocots and dicotyledonous (Cys in green beans, Cys gene in potato
gene, Cys genes in maize). Studies of the cystatin gene have been widely discussed focusing
on its relationship to resistance to drought, cold and salt etc.
With the above mentioned reasons and from the practical needs of groundnut breeding
optimism towards improving drought tolerance, we have carried out the thesis topic:

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"Creating drought tolerance lines and isolating cystatin genes related to drought
tolerance in peanut (Arachis hypogaea L.)”.
2. Research objectives

-

Create groundnut strain with drought tolerance higher than original breed using tissue
culture of plant cells.

-

Compare cystatin gene sequence of groundnut strain selected from scar tissue with that
of the peanut cultivars.

3.

Research contents

-

Screening scar tissue strain resistant to water loss affected by dry blowing and gamma
irradiation combined with dry blowing.

-

Analyzing the fluctuation of the number of selective lines through the generations.

-

Making comparison and defining the discrepancy in genome of the line selected by

RAPD technique.

-

Amplifying, separating and defining the sequence of the cystatin gene of selective
groundnut line and the original line.

4. New contribution of the topic
i) The thesis is a systematic research on application of technologies in plant cells to
improve and enhance drought tolerance of groundnut, from creating scar tissue, processing
scar tissues that create somatic mutation, selecting cell lines resistant to dehydration,
reviving plants, generating plants and analysis, assessment through generations, selecting
lines of groundnuts preeminent by drought tolerance and some biological and agricultural
features.
ii) Callus were treatment of scar tissue by gamma rays has been reduced in height
and rate of regeneration plants, change colors and leaf shapes. The findings are five
indicators specific RAPD two lines of contact selectively RM47, RM48: RM48/OPA07750bp;

RM48/OPA08-500bp;

RM48/OPB05-900bp;

RM48/UPC348-200bp;

RM47/OPH08-200bp. Discovery of peanut cystatin genes of group I phytocystatin, the most
closely related to cystatin green beans, farthest to the cystatin of kiwi fruit (42.9%). The
gene contains an intron and two exons coding for 98 amino acid protein. Cystatin of RM48
line derived from scar tissue is processed by gamma rays associated with the blower has 7
positions of amino acid differences compared to the same original L18.
iii) Determine the difference compared to the same original L18 on drought

tolerance of three lines lost RM48, RM47, R46 can be derived from scar tissue dehydration
is treated by gamma rays (2krad) with blower continuous 9 hours in in vitro culture systems.

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5. The meaning of scientific and practical
5.1. Scientific Significance
i) The results of the thesis provide scientific data to guide application of plant cells
to improve drought tolerance of groundnut. The technique selected cell lines under
dehydration and treatment of scar tissue to increase the frequency of mutations arising. The
approach and methods to evaluate the difference of the selected line compared to just touch
base in terms of morphology, expression yield component traits, drought tolerance, grain
characteristics and biochemical differences in the genome.
ii) Provide information on the cystatin and cystatin genes in peanut. Handling of
scar tissue by radiation combined with water is blown causing agent appeared cystatin gene
mutations as evidenced by differences in genetic sequences of the RM48 (derived from scar
tissue to be irradiated with blow dry) than the R46 line (derived from scar tissue be blown)
and like the original L18.
5.2. Practical significance
The results of a comprehensive review of drought tolerance of groundnut extent of
scar tissue, germinating seeds and young plants as a basis to assess and apply measures to
improve drought tolerance of groundnut. Results bred three communication lines derived
from scar tissue under treatment by irradiation and the blown advantage of drought
tolerance Vietnamese and some characteristics of agricultural biology, biochemistry, can
foster the seeds of new varieties or material for hybridization.
6. The structure of the thesis
The thesis consists of 128 pages (including references), divided into sections:

Introduction includes 3 pages; Chapter 1: Overview document, 34 pages; Chapter 2:
Materials and Methods, 14 pages; Chapter 3: Results and discussion Comment, 54 pages;
The conclusions and proposals: 2 pages; works were published concerning the thesis: 1
page. References: 15 pages; thesis has 22 tables, 20 pictures. There are 145 references in
Vietnamese and English.

Chapter 1. DOCUMENT OVERVIEW

The thesis has reference materials and review 34 domestic and 98 foreign documents
with related content, including: (1) Groundnuts and drought-resistant properties of the
peanut, (2) Advanced research drought tolerance of crops by plant cell technology, (3)

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Analysis and evaluation of selected lines derived from callus culture (4) Genes related to
drought tolerance in groundnut; (5) Cystatin and the role of cystatin in plants.
With the lead data collected, the analysis has confirmed that peanut valuable and
important position in the economy of many countries around the world. The trend of climate
change that changed the elements of environmental conditions and a growing number of
evaluation studies, breeding optimism towards higher resistance is made. Like other crops,
the methods used in breeding, including hybridization communication, breeding, mutant
selection from the population and use of modern biotechnology. The application of modern
techniques of biotechnology to improve plant resistance was conducted in two directions,
which are selected lines and transgenic somatic mutations. The success of gene transfer
techniques have been published on several crops and several properties related to the
drought tolerance of plants has improved.
The technique selected cell lines in higher plants based application understanding of

the omnipotence of the cell; heterogeneity of tissue or cultured cell populations; influence of
culture medium in forming a complete body ... make cultured cell populations can be
considered a population of plant cells, thereby screening the individual will be faster and
more efficient than conventional breeding methods applied on intact plants. Select the line
by increasing resistance to the unfavorable factors of the external environment has been
successful on several subjects such as rice, dry rice, wheat, tobacco ... These are our
suggestions for choosing in vitro culture techniques to improve drought resistance in peanut.
Drought tolerance of plants is due to multiple gene traits decision. Search trends
drought gene research is a major concern of many authors. The genes involved in drought
tolerance of groundnut published in recent years such as LEA genes related to cellular
dehydration. Su's research et al. (2010) on the same communication Luhua 14 detected at
least eight genes LEA. AhNCED gene encoding synthetic 9-cis epoxycarotenoid
dioxygenase was also confirmed related to the resistance of plants, the peanut AhNCED
isolated by Wan et al. (2005) size 2486bp, encoding a protein consists of 610 amino acids.
PLD gene encoding phospholipase D was also confirmed related term was found in two
types of communication is through AhPLD1 and AhPLD2 Nakazwa team (2006) ... Cystatin
phytocystatin in plants is called, consists of two groups, different in size, mass and areas
associated with the cysteine proteinase. Since 1987, cystatin genes in rice were isolated, the
cystatin gene has been isolated in many plants, but the peanut was little known. Study the
function of cystatin, many authors discuss the relationship of cystatin with tolerance of
plants. The complexity of the structure of the cystatin genes, the ability of gene expression
in different growth stages of crops and in relation to the resistance of the disadvantages of

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the external ... Some published on gene expression of cystatin-related drought tolerance has
been demonstrated cove on bean plants, vegetables genes, sea mustard, winter wheat ... is

the basis for our continued search and analysis on peanut cystatin genes.

Chapter 2. MATERIAL AND METHODS

2.1 Plant materials
The thesis using 10 peanuts cultivars as the materials research. In particular, 8
varieties (L05, L16, L18, L23, L24, V79, MD7, MD9) offered by the Center for research
and development Legum, Institute of Food Crops Vietnam. SD30 variety provided by
Center of Agricultural Extention Nam Dinh province and red peanut (red BG) due to Bean
Development Center Legum Viet Yen district, Bac Giang Province has to offer.
2.2 Chemicals and equipment
Using the chemical purity and dedicated derived from reputable companies such as
the drug company's growth Sigma; Taq-polymerase, EDTA, SDS, agarose ... Invitrogen's.
2.3. Research methodology
2.3.1. Cultured in vitro method
To create the technical communications in vitro culture, the culture of the steps:
creating scar tissue completely in the dark (10 days) to conduct the airflow caused by
dehydration of the box sterile culture and irradiated and then combined to cause dehydration
by blowing dry recycling plant, creating a complete tree as described by Nguyen Thi Tam et
al. (2006).
2.3.2. Field research methodology
Mark individual plant lines regenerated from callus. To make the planting and care as
directed by the Ministry of Agriculture and Rural Development. Track indexes and
agronomic performance of plants at maturity.
2.3.3. Method of physiological, biochemical
Quantification of soluble protein by the method of Lowry; quantitative lipid Soxhlet amylase activity by the method of Heinkel; The methodαmethod; Define of sugar analysis.
Assessing dehydration tolerance of scar tissue by staining of cells et al. Towill (1975).
Assessing drought-resistant seed germination stage method caused due to sorbitol -amylase,
sugar.α7% defined tolerance by determining the activity of Assessing drought tolerance at
seedling stage by artificial methods to cause limited by Le Tran Binh et al. (1998).

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2.3.3. Methods in molecular biology
Total DNA extraction method Gawell et al. (1991). DNA polymorphism analysis by
RAPD technique. Isolation of genes by PCR technique. Separation of the gene by the
method of Sambrook et al. (2001). Gene sequence was determined on the ABI PRISM 3100
Avant automatic Analizer Genetic, Institute of Biotechnology.
2.3.4. Methods of data analysis
Analysis of statistics with Excel software as Chu Hoang Mau (2008) Data analysis by
RAPD NTSYSpc software version 2.0; Results of genetic analysis using BioEdit and
DNAstar

software.

2.4. Study location
The experiment was conducted at the University of Pedagogy; University of Science;
Institute of Life Sciences-University of Thai Nguyen.
Irradiation gamma ray irradiation at the National Center, Tu Liem, Hanoi.
DNA sequences were determined at the Institute of Biotechnology.
Field experiments are located at the Forest cane, Quang Vinh Ward-Thai Nguyen
city.

Chapter 3. RESULTS AND DISCUSSION

3.1. RESULTS CREATED THE DROUGHT TOLERANCE BY TECHNICAL
CALLUS TREATMENT IN THE IN VITRO CULTURED SYSTEM

3.1.1. Screening lines callus subjject blow dry
3.1.1.1. Ability to create scar tissue and the growth of scar tissue studied 10 breeds
optimism
For the purpose of assessing scar tissue of the same communication in in vitro culture
systems as the basis for the research to create drought-tolerant line of communication with
the plant cell technology, we surveyed the ability to create scar tissue from the embryo of
the seed cells and speed the growth of scar tissue in 10 varieties studied communications.
Research results show that rate of scar tissue from embryos of the 10 varieties ranged from
82.71% optimistic (L18) to 98.55% (V79). The same communication rate of scar tissue over
the same 97% MD7, MD9, V79. Same can create scar tissue ratio lower than 90% include
the same L18, L23, Red BG. In particular, the proportion of scar tissue created at least the
same L18 (82.71%). The volume of the same communication scar tissue is formed ranged

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from 115.00 mg (L18) to 198.00 mg (V79). The same L05, L23, MD7, MD9, SD30, V79
mass of scar tissue over 180mg. Four varieties L16, L18, L24 and Red BG mass of scar
tissue formed by more than 160 mg. The results showed the ability to meet the growth of
scar tissue and scar tissue of the 10 varieties of communication, be sure to use for
subsequent studies in selected cell lines.
3.1.1.2. Dehydration tolerance of scar tissue the same communication research
Dehydration tolerance of scar tissue is determined by the dehydration, the results of
rapid assessment of scar tissue viability by TTC staining method and determine the survival
of scar tissue after treatment by blowing dry. The results determine the survival rate of scar
tissue after treatment with blown shown the stamina of cells and this is the basis for
screening cell lines. The scar tissue dehydration under which we obtained the materials to
make recycling plant for selecting drought-tolerant line of communication.

Results of studies of tree regeneration under dehydration scar tissue that, like us all
participated in the study were able to regenerate plants from callus survived. The models for
survival rate from 83.33% tree reborn to 100.00%. Based on research and results, we choose
the time scale of 84% water loss from the initial fresh weight and tissue survival rate of
about 10% to 20% is the threshold to filter the line.
Scar tissue processing and technical communications dry wind scar tissue, we
identified peanut varieties L18 resistant dehydration and choose the best cream puff dry 9
hours threshold to filter. Results of assessment of dehydration tolerance of scar tissue,
associated with evaluated tolerance of 10 seed lost in the period when the seed germinates
and physiological drought period when young trees have shown that artificial limit , in 10
varieties studied communications, L18 resistant varieties dehydration least, have - amylase
and sugar content in the lowest group inαthe activity of the day when the seed germinates
term, the lowest drought index . This result is consistent with published by the Center for
beans, Institute of Food crops and trees provide food Vietnam.
3.1.2. The effect of gamma rays associated with dry wind to the survival and
regeneration of plants like communications L18
Like peanut varieties L18 is defined as strains resistant to drought, low water loss
under dehydration among 10 varieties of research communications. In addition, L18 is the
same high-yield varieties. So, with the goal of improving drought tolerance of the varieties
have lost tolerance cream, we conducted surveys combined effects of gamma irradiation for
the subject screening dehydration using the technique for blowing dry peanut varieties L18
with scar tissue.

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Results exploring the effects of gamma-rays are performed on the fifth dose level
radiation: 0.5 krad; 1.0 krad; 2.0 krad; 3.0 krad; 4.0 krad blown with scar tissue in 9 hours .

The results determine the rate of tissue regeneration of tree survival showed regeneration of
scar tissue plants affected by irradiation combined with blown ranged from 21,30% to
88,87%. Compared to the regeneration of plant tissue to survive only be blown, then the
process of scar tissue by irradiation with blower reduces the rate of tree regeneration.
Significant difference of plant regeneration is affected by irradiation combined with the
blower that is, tree regeneration of a change in morphology, particularly, variation in color
and leaf morphology.
With low-dose irradiation (0.5 krad; 1krad; 2krad), the leaves are still green as the
tree control. But with high-dose irradiation as 4krad 3krad and greens of the leaves appear
in about 1-2 weeks after the leaves begin to yellow UA. Small leaf morphology and leaf
curling occurs. Plant growth speed is inversely proportional to the intensity of the radiation
dose. At higher doses of radiation phenomena plants gradually died, relatives barren.
Regenerating tree height is much lower than the control plants.
Table 3.6. The effect of irradiation combined with blower 9 am to survival and regeneration
of tissue in the same communication tree L18
Radiation dose
combination
blower
9 hours

Rate of
survived
tissue
(% )

Rate of
regeneration
(% )

0,5krad

34,23 ± 0,37

47,63 ± 2,37

Normal

1,0krad

33,65 ± 0,52

88,87 ± 5,57

Normal

2,0krad

28,42 ± 0,43

38,87 ± 5,54

Normal

3,0krad

18,87 ± 0,59

26,17 ± 4,97

Small, yellow and fallen leaves,

curly leaves edges

4,0krad

12,51 ± 0,42

21,30 ± 6,03

Small, yellow and fallen leaves,
curly leaves edges

Patterns of regenerated trees

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A

B

C

E

D

G

Figure 3.3. Some pictures of the same tree regeneration touch L18
6-week-old stage
A: Plant regeneration not blown;

B: Plant reproduction under blower 9 hours;

C: irradiation dose 0.5 krad + blower 9 hours

D: irradiation dose 2.0 krad + blower 9 hours;

E: irradiation dose 3.0 krad + blower 9 hours;

G: irradiation dose 4.0 krad + blower 9 hours

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From the results of the evaluation survival and regeneration of scar tissue scar tissue
after treatment with gamma irradiation we have identified the critical dose of scar tissue
radiation dose 4krad la communication. If you exceed the limit 4krad most dead scar tissue,
can not produce enough of the line model for the further research, especially research on the
selection line. However, to obtain multiple mutations need to determine the appropriate dose
of irradiation, to have created the beneficial genetic variation, both the rate of the tissue
regeneration and high survival. Therefore, we selected the dose 2krad blower with 9 hours
to screen the tissue and the entire plant.
Result of treatment of scar tissue in in vitro culture systems of peanut varieties L18
continuously blown by 9 hours and radiation dose in combined blown 2krad continuous 9

hours were obtained from 167 current and measuring 198 models communication lines are
created.
3.1.3. Agro-biological characteristics of the communication line in R0 and RM0
The study population lost R0 (plants regenerated from callus resistant blower), RM0
(Plant regeneration from callus under irradiation with blow dry) found that the
communication line from the test tube to grow in the field (spring, 2008) that genetic
variation is much larger than the original species in all study criteria (Table 3.7).

Table 3.7. Agronomic characteristics of the population selected communication R0, RM0
( X : Mean value; S X : average deviation form; Cv%: coefficient of variation)
Target tracking

Original var.

R0 Population

RM0 Population

Main
body X ± S X
height (cm)
Cv %

37,00 ± 0,58

57,00 ± 4,61

18,50 ± 1,91

2,70

25,58

10,32

Number
of
branches/ plant

X ± SX

8,33 ± 0,33

3,00 ± 0,39

6,75 ± 0,57

( branches)

Cv %

6,93

41,57

8,38

Number
of X ± S
X

fruit/ tree plant
Cv %
( fruit)

20,67 ± 0,67

22,80 ± 1,81

14,00 ± 1,96

5,59

25,05

13,98

X ± SX

16,33 ± 0,33

14,80 ± 1,50

8,17 ± 1,36

79,00

64,91

54,65

Số
quả
chắc/cây
Tỷ lệ quả chắc

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As such. the process of selecting the line lost drought include: (1) Create callusand scar
tissue; (2) regeneration of trees and Tree Planting complete (3). track and analyze the genetic lines
of selectivity.
Scar tissue in the peanut processing plant in vitro culture, selective survival of scar tissue,
regenerating plants and monitoring populations and RM0 R0 can draw some comments below:
(1) The line comes from scar tissue dry wind bear up tree height than the original species, while the
line is derived from scar tissue in combination with radiation suffered blow dry reduced plant height
compared to the same original.
(2) Populations of plants produced by tissue culture techniques of plant cells have some quick /
plant than it resembles the original. Military R0 can quickly reach 3 / tree, populations reached 6,75
RM0 fast / tree, like the original with rapid 8,33 / plant.
(3) Population R0 is 22,80 fruit / tree, more than the same root (reached 110.30% from the same
root); population RM0, with some troops may fruit / tree less like the original, only 14.00 fruit / tree
(67.73% from the same root).
(4) Coefficient of variation for genetic research targets of the two forces can be created by plant
tissue culture techniques of plant cells larger than the original species.
From the evaluation results in generation of R0, RM0, we chose the 7 line is derived from the
same communication L18, in which three lines originating from the scar tissue is influenced by the
blower is R44, R46, R48 and 4 lines derived from scar tissue affected by radiation combined with
blower is RM46, RM47, RM48, RM49 a starting material to review and filtering in the next

generation.

3.2. RESULT ANALYSIS OF SELECTED LINES THROUGH THE GENERATION
3.2.1. Assess the stability of agricultural biological characteristics of the selected
contact in the first generation, the third generation.
Compared to other crops, peanuts are difficult maintenance, and low germination
when stored last time. Number of fruit trees in the first generation touch is not large enough
to carry out research related to tolerance. Therefore, the 7 whole grains in the military can
communicate selectively R0, RM0 we look into the service next year looks. Results
assessing the stability of agronomic traits on the main stem height, number of branches /
plant, number of fruits / plant, number of fruits make / tree generation R1, RM1 look at
winter harvest in 2008 (Table 3.8 ); generation R3, RM3 look at winter harvest in 2009
(Table 3.9) showed the stability of agricultural biological characteristics of the
communication line is derived from the scar tissue has contributed to confirm the value of
carefully Arts tissue culture plant cells could create new strains and shortening breeding,
increasing the efficiency of the process of breeding.

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3.2.2. Evaluation of selected lines in the fifth generation
We assess the current selection in Thursday generation through the analysis of a
number of state and other biochemical indicators; assess drought tolerance in seed
germination stage, seedling stage and differences in genome by RAPD technique.
3.2.2.1. Agronomic traits, grain yield and quality of the communication line choice in the
fifth generation
Results evaluation of stability of agronomic traits of selective line 7 in the fifth
generation Fall Winter crops 2010 shows contrition. biological agriculture norms are the

fluctuations in the low range 1,66% to 7,23%. We conduct assessments and analyses of the
characteristics. quality grain yield of 7 lines selected fifth generation through identifying
elements. such as productivity. mass volume 100pods; 100 seeds; quality assessment on
aspects of Biochemistry through identifying a protein, lipid content. Results of research on
biological characteristics of agriculture. the quality of the contacts selected derived from
callusis dehydrated and callusinfluenced irradiation combined with handle causing
dehydration. we selected 3 lines has outstanding features, including: (1) The R46 has a mass
of 100 fruit and seeds; (2) Lines of RM47 protein content higher; (3) Line of RM48 lipid
content higher.
3.2.2.2. Results evaluated tolerance of the selective in the fifth generation
Drought tolerance of plants in general and in particular peanut is a trait difficult to
control in the field. Therefore, we evaluated the tolerance of the current generation of
communication selected fifth in seed germination stage and seedling stage.
Causing the current term optimism fifth selective generation seed germination stage
by treating the seeds with 7% sorbitol, and then α-amylase and determine drought tolerance
through the activity of sugar. The results showed that the -amylase and sugar content of the
seeds germinated andαactivity of tended to increase from 3 to 7 days old days old and then
decreased in stage nine days of age (Figure 3.5 and 3.6).
Analysis of correlation between the variation of α-amylase activity and sugar content
of communication like L18, L23 and the line selected in the period when the seed
germinates physiological limits, the results presented in table 3.11.
Table 3.11 shows that the α-amylase activity, sugar depends linearly on correlation
coefficient (R) ranged from 98,11% to 99,99%. The α -amylaseα activity of high resolution
will make the process of starch to sugar occurs powerful, guaranteed to provide nutrients for
the germination of seeds, especially the adjustment of osmotic pressure in cells extreme

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conditions. The -amylase and the way of the line, likeαdifference in the activity of us at the
time of seed germination vary regarding their drought tolerance.
2

0,7

1,8
1,6

0,5

% hạt nảy mầm

ĐVH Đ/m g h ạt nảy m ầm

0,6

0,4
0,3
0,2
0,1

1,4
1,2
1
0,8
0,6
0,4

0
3

5

7

9

0,2

Ngày tuổi nảy m ầm

0
R44

R44

R46

R48

RM46

RM48

RM49

L18

L23

R46

R48

RM46

RM47 RM48

RM49

L18

L23

RM47
3 ngày

Figure 3.5. Variations on α-amylase
activity of the touch like L18, L23,
and the selection of limited
physiological conditions

5 ngày

7 ngày

9 ngày

Figure 3.6. Fluctuations of similar
sugar lost L18, L23 and the limited
selection in terms of physiological

Table 3.11. The correlation between the activity of α-amylase and sugar content of
communication like L18, L23 and the selection stage seed germination
the Regression equation

No

Varieties
selection

1

R44

1,13x + 0,72

0,9881

2

R46

1,61x + 0,57

0,8972

3

R48

1,04x + 0,77

0,8379

4

RM46

1,83x + 0,34

0,9811

5

RM47

0,54x + 1,04

0,9945

6

RM48

2,66x + 0,66

0,9999

7

RM49

1,04x + 0,68

0,9862

8

L18

1,07x +0,70

0,8466

9

L23

2,26x + 0,19

0,9983

and

Correlation

Assessing drought tolerance at seedling stage of the selection fifth generation by

determining the rate of withered tree, the tree recovered in the first 5 days time limit and
determine the relative drought indices for. RM48 line with the highest drought index

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(10,089.20%), then to the R46 and L23 cultivar. At least the current R44, RM49, and like
the original L18. In the seventh line of communication like the original selection and L18
have 5 lines selected drought index is relatively high, including two lines derived from
callus resistant blower (R46; R48) and three lines derived from tissue scar affected by
irradiation combined with a blower (RM46; RM47; RM48).
3.2.2.3. Assessing the differences between the genomes of the communication techniques
selected by RAPD
The results of analysis with 25 primers RAPD reaction randomly collected a total of
1254 segments, estimate the size of segments ranging from 0,2 kb to 4,2 kb. Total segment
polymorphisms as 255 segments, accounting for 20.33% of the segment to be cloned. The
results compare differences in rates at the molecular level between the genomes of the
selected pair and like the original show, 8 gene system error rate from 1,521% to 9,143%.
The level is the biggest difference dong dong RM48 and R48 (rate difference of 9,143%).
Figure 3.10 describes the relationship of the seven original species and the selection at the
molecular level with 25 random primers in RAPD reactions.
L18
RM46
R44
R46
RM47
RM49

Figure 3.10. Diagram describes the relationship of seven lines with similar selection at the
molecular level the original L18 with 25 random primers

The results in Figure 3.10 shows the selected communication line 7 and the same
original L18 are classified into two groups with genetic distance is 12%. Group I: There is
only one line of RM48, which is derived from the scar tissue affected by a combination of
radiation and blow dry, with genetic distance from the line and just rest in branch 2 is 22%.
Group II: consists of the R48, RM49, RM47, R46, R44, RM46, and like the original L18.
Genetic distance of the original species selection and L18 is 9.5% (1-.905 = .095). Group II
is divided into two branches, first branch is the original species (L18); second branch, is
divided into several small branches of the remaining selection, R44 and R46 along two lines
originating from the scar tissue under blowing dry, which differs from the original species
with a low 1.521% and 1.522%.

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Coefficient of genetic diversity (HRAPD) of seven selected communication line within
the 25 primers used at the molecular level were identified: HRAPD = 11.09%. Have
identified five molecular indicators RAPD featured in two communication lines with 5
primers selected randomly appear RM48 line features four sizes with four primers. In
particular, specific primers OPA07 size appears at 750bp (RM48/OPA07-750bp) primer
OPA08 size characteristics appear at 500bp (RM48/OPA08-500bp) primers specific OPB05
size at 900bp (RM48 / OPB05-900bp) specific primers UPC348 size appears at 200bp
(RM48/UPC348-200bp). OPH08 prey appears a characteristic 250bp in size with the RM47
(RM47/OPH08-200bp). Gather the results analysis of the characteristics of agricultural
biology. seed quality. resistant and molecular biology characteristics of selective line
derived from callusis blow dry and callusis irradiated combined with blow dry breeds L18 is

presented in ficture 3.11 and table 3.16
Table 3.16. Some characteristics of selective lines and variety L18
Characteristic

RM48

RM47

R46

L18

Plant
Regenerate
from calli
treatment with
irradiated+ dry
blow

Plant
Regenerate
from calli
treatment with
irradiated+ dry
blow

Plant
Regenerate
from calli
treatment with

dry blow

Imported from
China

Plant height (cm)

35,46 ± 1,02

41,63 ± 0,54

45,77 ± 0,79

30,62 ± 0,84

Weight of 100
pods (g)

108,27 ± 6,98

103,43 ± 4,46

145,12 ± 5,05

116,59 ± 2,84

Weight of 100
seed (g)

46,53 ± 2,89

41,11 ± 2,50

56,27 ± 1,32

47,81 ± 0,55

Protein Content
(% KLK)

27,72 ± 1,96

38,97 ± 4,25

32,08 ± 2,54

34,47 ± 2,85

38,67 ± 0,88

36,89 ± 0,89

34,67 ± 0,77

36,00 ± 2,67

10089,20

5272,80

7662,38

4725,09

Source

Lipid content
(% KLK)
Drough Index
Specific Primer
RAPD

RM48/OPA07-750bp;

RM48/OPA08-500bp;

RM48/OPB05-900bp;

RM48/UPC348-200bp;

RM47/OPH08-250bp;

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RM48

RM48

RM47

RM47

R46

R46

L18

L18

Figure 3.11. Images of fruits and seeds communication lines selectively
earnings at fifth generation, just like touch touch L18 and L23

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3.3. ISOLATION AND SEQUENCING GENES CYSTATIN FROM PEANUTS
3.3.1. Amplified DNA genomes of cystatin genes from peanuts
After the extraction of total DNA. glass cleaner and headlight on the
appropriate concentrations. we have conducted gene cloned from DNA by PCR
cystatin genomes of seven lines of seed selection. L18. and same L23. Human genes
are check result on 1% agarose gel with marker (Figure 3.12).
L23 RM49 RM48 RM47 RM46 R48 R46 R44 L18

M

Figure 3.11. Photos electrophoresis products of the same human cystatin gene L18,
L23 and 7 line research communicationh

PCR reaction product obtained in Figure 3.11 shows, only one DNA band,
approximately 500bp in size, consistent with theoretical calculations on the size of
cystatin genes that Yan et al. (2004) when This gene was isolated from mRNA.
Therefore, we believe that it is the result of PCR with primer pairs-AraF Cys / CysArar. Cystatin gene was cloned from the DNA like us L18, L23 and 7 selective
communication line near 500 bp in size. However, to affirm correctly that cystatin
genes, we performed separate line, identifying the sequence comparison with cystatin
gene sequence was published.
We perform a separate line and determine the order of 2 cystatin genes dong
R46, RM48 (two lines have the highest tolerance), similar to the original L18
(drought-resistant cream) and similar to L23 (with tolerance good) for comparison
purposes cystatin gene sequence of lines and varieties.
3.3.2. Results of cloning and sequencing of genes cystatin
Result of splitting the colonies carrying cystatin genes were tested by clonyPCR reaction. Defining the nucleotide sequence obtained from the plasmid of the
sample is treated with DNAstar software shows that cystatin genes of the same
communication L18, L23, R46 and RM48 lines are 461 nucleotides in size.
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Comparison of sequences with nucleotide sequences isolated from cDNA of 2 cystatin
genes in plant gene bank communication on international codes AY722693 is the
sequence used to design primers and sequence bearing codes EU723567, we get that
the cystatin genes encoding proteins that we isolated, including two exons and one
intron. The first exon of the gene segment of 102 nucleotides starting from position 1

to 102 and the second exon 195 nucleotides, from position 267 to 461 zone in the
middle of a 164 nucleotide intron from position 103 to 266.
3.3.3. Results comparing the gene sequence and protein cystatin
3.3.3.1. Comparison of nucleotide sequences of the cystatin gene
Results comparison of nucleotide sequences of the same tribe of cystatin genes 2
L23 with two lines touch L18. R46. RM48 is created by the technology of plant cell
tissue cultured has the wrong. In particular. the sequence of nucleotides and the R46
L18 varieties derived from callussuffered dehydration is the same entirely. The wrong
nucleotide position RM48 has 19 different than the same root (L18). Cystatin-like
tribe's genome Nucleotide L18 has 14 different compared to the same position wrong
L23.
Cystatin genes of leguminous green relatively conservative. there is no difference
in terms of genes of drought well and cystatin-like drought worse. Results identified
the nucleotide difference cystatin genes peanuts (table 3.17) shows the difference of
cystatin genes appear in both the Club and the intron exon. In exon 1. just the wrong
positions L18 has 2 L23 and 12 other wrong locations other than the RM48. In exon 2.
just the wrong positions L18 has four different varieties of L23 and had a wrong
locations other than with the RM48. In addition. cystatin genes and RM 48 breeds
there are 11 L23 position discrepancy lies in the intron. As such. is derived from the
same L18. two lines touch the wrong selection can vary in the sequence of nucleotides
of cystatin genes. and the difference between the same expression is likely to be poor
(L18) with the same term is likely to be a better term (L23). Research results
demonstrate. effective processing gamma rays associated with blow dry raises cystatin
gene mutation frequency in comparison with callusis only liable to blow dry.
3.3.2.2. Comparison of nucleotide sequences in the coding region
Because cystatin gene isolated from DNA containing the intron of
communication does not exist in the process of translation, we proceed to remove the
pairing of the intron and exon, the result is the 297 nucleotide long encoding cystatin.
Compared with the 6 protein sequence of the gene segment encoding cystatin of plant
sizes, respectively published in the international gene banks.

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Results evaluated through analysis of level differences, similarity level (Table
3.18) found four genes encoding the order disrupted communication co cystatin levels
similar to cystatin 6 the order of other crops published in gene bank international, from
45.9% to 100%. Nucleotide sequence similarity of peanut-Definition from 98.0% to
100%. Especially, do not have any differences on any of the same nucleotide
selectively communicate with the L18 and R46 of the same nucleotide sequence
Arachis hypogaea L. on the international gene bank (code AY722693).
Establishing the genetic relationships of the gene encoding cystatin of 10
sequences, the relationship diagram of the gene encoding cystatin be classified into
two groups: Group I sub-divided into three large groups and small groups of women
includes the same communications and other crops, including communications lines
and similar distribution in the same group. Group and the same communication lines
(R46, RM48, L18, L23, AY722693) is closely related to cystatin with green beans
(number AM712476), then the seed of rice cystatin-number S49967, barley and riceY12068 -AB125973. Group II is the longest distance to the remaining group and only
one species is kiwi fruit (AY390352), error rate is different than the peanut plant
cystatin was 41.5%.
Table 3.18. High level similarities and differences in the coding sequences of genes
Similarity level
1.L18 cultivar
2. R46 line
3. RM48 line
4. L23 cultivar
5. Arachis hypogaea
(code AY722693)
6. Oryza sativar

(code: S49967)
7. Actinidia deliciosa
(code AY390352)
8. O. sativar
japonica (code
AB125973)
9. Vigna radiata
(code: AM721476)
differences level
10. Wheat (code
Y12068)

3.3.2.3. Comparison of amino acid sequence of the protein cystatin
Nucleotide sequence of the cystatin gene encoding the same communication
L23, L18 and two communication lines R46, RM48 was translated by software code to
create molecular DNAstar of 98 amino acid protein, with a code beginning with AUG
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provisions of methionine (M) and the code ending UAA. Results comparing the order
of research with four specialized software BioEdit represented by Figure 3.16.
Comparison of amino acid sequence of the communication research, we found
that all four amino acid sequence contained two regions of the group is conservative
and QVVAG LARFAV. The specific locations of the two segments are conservative:
L22A23R24F25A26V27 and Q49V50V51A52G53. Compared with the classification
of Margis et al. cystatin gene (2008) and Martinez et al. (2008), we found cystatin
genes of the same communication and research under the category of phytocystatin 1.

Figure 3.16. Comparison of amino acid sequences of the four sample
Combined with the results of genetic analysis, research results show that the
location of glutamine 34 (Q34) and asparagine 35 (N35) is a pair of amino acids
encoded by the first three exon junction 2 of the cystatin . Position corresponding to
position it as arginine 34 (R34) and asparagine 35 (N35) of cystatin RM48 line.
Glutamine and arginine at position 34 is two different amino acid structure
characteristics of the original R, in which glutamine is the amide of glutamic acid;
different from the amino acid arginine amino acid control group. This difference is
related to molecular structure and activity of cystatin in the inhibition of cysteine
proteinase activity, is suggested for further study of cystatin in groundnut.
Besides differences in amino acid position 34, resulting in amino acid sequence
comparison of the same communication L23, L18 and R46 2 lines, RM48 we found
more variation in the position 6 amino acid sequence, bringing the total number of
amino acid changes in the cystatin touch location is 7. Specific differences shown in
position 29, 30, 31, 32, 33, 34 and 36 of the line and the same study (Figure 3.16).
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If transmission of the same cystatin L18 compare la base, we found that, similar
to peanut L23 has a different position is wrong amino acid position 36, alanine
replaces glycine. The biggest difference of cystatin cystatin of the same
communication line L18 is RM48 (7 other wrong position). Analyze each other related
amino acid sequences of lines, the same analysis with different level of drought
indicates that, similar to L23 has good drought tolerance, the error is different from the
sequence of drought-cream group in position tri 36, cystatin RM48 line now also have
differences. However, do not find every relevant variation of the amino acid at
position

29,

30,

31,

32,

33,

34

RM48

line

with

the

order

studied.

The results of analysis of cystatin amino acid components like lost L23, L18 and R46
selected second communication line, RM48 showed significant differences on the
amino acid and the same communication lines. However, the statistical results the total
number of amino acid hydrophilic, hydrophobic or group of amino acids essential for
people who do not receive the difference between lines and selective breeding. The
amount of hydrophobic amino acid occupies a relatively large proportion of cystatin

communication component (45.92%) allows predicting the residence of the cystatinrelated drought tolerance of peanut plants. Clear but will distributed hydrophobic
protein on the cell membrane or system offers, the preferred water-soluble protein
primary translation. Therefore, with functional cysteine proteinase inhibitor activity
can be very optimistic participants cystatin protect trees bearing cells will be effective
in conditions of extreme water shortage.
CONCLUSIONS AND RECOMMENDATIONS
1. Scar tissue processor communications bandwidth of 10 similar techniques blower
continuously for 3 hours, 6 hours, 9 hours and 11 hours were determined to be the
same optimistic L18 tolerant low water. At the threshold selected nine hours of
irradiation with gamma rays 2krad reduced rate of recycling plants and the phenotype
appears

less

in

lost

crops.

2. Regeneration communication line 198 is derived from scar tissue and scar tissue
under dehydration under irradiation with gamma rays to cause dehydration, analyzed
through five generations have selected three communication lines (RM48, R46,
RM47) can high drought tolerance from drought-tolerant varieties less optimistic L18.
3. Three lines are not selective about the advantages Vietnamese drought tolerance, but
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also changes in the phenotype: The RM48 is the highest lipid content (38.22% KLK),
a genetic distance compared to the same line and Original is 22%. RM47 Series has
the highest protein content (38.97% KLK). R46 Series volume 100 results (145.12 g),
100 grains (56.27 g) was highest. Two lines R46 RM47 and genetic distance for L18 is
9.5% of original breeds. Dong RM48 refreshing sound promising new varieties.
4. Have determined the coefficient of genetic diversity of the selected communication
line is HRAPD = 11.09%. There are five unique RAPD indicators identified in the two
communication lines: lines appear RM48 4 indicator featuring four new sizes:
RM48/OPA07-750bp; RM48/OPA08-500bp; RM48/OPB05-900bp; RM48/UPC348200bp), the RM47 features OPH08 primer 200bp in size (RM47/OPH08-200bp).
5. Amplification and separation of the cystatin gene from DNA into the genome of a
selected communication line and the same root. Cystatin gene of peanut has 461
nucleotides, with two exons and one intron, the first group of phytocystatin, the most
closely related to cystatin green beans, farthest to the cystatin of kiwi fruit (42.9%).
The protein encoded by the gene has 98 amino acid, including 45.92% hydrophobic
amino acid, amino acid 54.08% and 28.57% hydrophobic amino acid essential to the
total

amino

acid

protein

of

cystatin.

6. Compare the order of the rows of peanut protein cystatin select and breed seeds
have been detected in seven amino acid changes, including co dong RM48 biggest
change. This has affirmed the process of scar tissue irradiated by gamma rays have

done major changes in the peanut genome structure leads to a change of phenotypic
larger than just the scar tissue processed by the dry wind.
RECOMMENDATION

1. Continue to monitor, analyze and communicate the advantages refreshing
Vietnamese 3 is R46, RM47, RM48 to introduce assaying.
2. Design vector carrying cystatin gene and moved on peanuts and other crops as well
as to study the effects of cystatin drought resistant and some other characteristics in
plants.

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PUBLICATIONS

1. Vu Thi Thu Thuy, Nguyen Thi Tam, Chu Hoang Mau (2009), Selection for
dehydration tolerance in vitro of some peanut cultivars, Science and technology
journal of Agriculture & Rural development, 7: 14-19.
2. Vu Thi Thu Thuy, Nguyen Vu Thanh Thanh, Chu Hoang Mau, Nguyen Thi
Tam (2009), Analysis of cystatin gene sequenceing of L18 peanut cultivar
(Arachis hypogaea L.), Báo cáo Hội nghị Sinh học toàn quốc: 397-400.
3. Vu Thi Thu Thuy, Dinh Tien Dung, Nguyen Thi Tam, Chu Hoang Mau
(2010), The results of selection of some peanut lines from losing water scar tisue
from L18 cultivar, Journal of science and technology, Thai Nguyen University,
72(10):122-126.
3. Vu Thi Thu Thuy, Nguyen Thi Tam, Chu Hoang Mau, Nguyen Vu Thanh
Thanh (2011), Characteristics of nucleotide sequence of cystatin gene of some
peanut lines derived from irradiated and dehydrated, Journal of Biology, 33(1):

86-95.
5. Vu Thi Thu Thuy, Nguyen Thi Tam, Chu Hoang Mau, Nguyen Vu Thanh
Thanh (2011), The selection of the variational lines which resistant to
dehydration and radiation of peanut crop (Arachis hypogaea L), Journal of
Biotechnology: 9(3): 1-8.

6. Submistion 04 cystatin gene sequence on NCBI
(1) Vu,T.T.T., Nguyen,T.V.T., Chu,M.H. and Nguyen,T.T. (2010), Arachis
hypogaea cystatin gene 1, exons 1-2, EMBL, GenBank, Accession FN811133.
(2) Vu,T.T.T., Chu,M.H., Nguyen,T.T.V. and Nguyen,T.T. (2010), Arachis
hypogaea cystatin gene for cystein proteinase inhibitor, cultivar L23, EMBL,
GenBank, Accession FR691053.
(3) Vu,T.T.T., Nguyen,T.T., Chu,M.H. and Nguyen,T.T.V. (2010), Arachis
hypogaea cystatin gene for cystein proteinase inhibitor, cultivar L18, EMBL,
GenBank, Accession FR745399.
(4) Vu,T.T.T., Nguyen,T.T., Chu,M.H. and Nguyen,T.T.V. (2011), Arachis
hypogaea partial cystatin gene for cystein proteinase inhibitor, cultivar L18,
EMBL, GenBank, Accession FR828803.
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The work was completed at the Department of Genetics & Biology Modern
Biology-Agricultural Engineering, University of Pedagogy-Thai Nguyen
University

Science Instructor:

Prof. Dr. Chu Hoang Mau

Prof. Dr. Nguyen Thi Tam

Objection 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.....................................
Objection 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.....................................
Objection 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.....................................

The thesis will be protected before the Board level thesis dots
Meeting at the University of Pedagogy-Thai Nguyen University

At last ...... hours, days ...... May 2011

Thesis can be found at:
- National Library
- Learning Resource Center Thai Nguyen University
- Library Pedagogical University, Thai Nguyen
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