(Luận văn) map based cloning of leaf rolled inside (lri) mutation in arabidopsis thaliana

Combined with the results of An et al 2014 suggesting that mutant gene, At2g32460 caused leaf rolled inside phenotype in AP-44-1 mutant line Key word Map-based cloning, leaf rolled insi

Trang 1

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

NGUYEN THU HUONG

MAP - BASED CLONINGOF LEAF-ROLLED INSIDE (LRI) MUTATION IN

Trang 2

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

NGUYEN THU HUONG

MAP - BASED CLONINGOF LEAF-ROLLED INSIDE (LRI) MUTATION IN

Supervisors: Prof Park Soon Ki

PhD Pham Bang Phuong _

luan van tot nghiep download luanvanfull moi nhat z z @gmail.com Luan van thac si

Trang 3

Supervisor (s) Prof Park Soon Ki

PhD Pham Bang Phuong

Map-based cloning is the method of identifying a mutation gene by looking for

linkage to markers that physical location in the genome Mutant line, named AP-44-1,

to find a mutation gene, mapping population was conducted and phenotypic analyzed using F2 plants of mapping lines Genomic DNA was prepared from leaves of 689 F2plants for analysis In order to define chromosomal region where mutation located, PCR-based mapping was performed using SSLP molecular markers Finally, the region containing mutant gene was narrowed down to an approximately 163kb Candidate genes in an interval of ~163kb were sequenced to identify the mutant gene

Combined with the results of An et al (2014) suggesting that mutant gene, At2g32460 caused leaf rolled inside phenotype in AP-44-1 mutant line

Key word Map-based cloning, leaf rolled inside, At2g32460, AP-44-1

Number of

Date of Submission 8/2016

Trang 4

ACKNOWLEDGEMENT

First and foremost I would like to express my sincere gratitude to Prof Park Soon Ki and Dr Oh Sung Aeong for all the guidance, helpful advice during the whole period

I would like to give a big thank to Dr Nguyen Tien Dung and MSc Nguyen Thi Hoai Thuong for their valuable suggestions and critical review of my thesis

I would like to thank Faculty of Biotechnology and Food Technology members for their support through my internship

I also would like to thank PhD Pham Bang Phuong for helpful advice during all this time

In addition, I would like to thank all members in Sexual Plant Reproduction Laboratory for their help

Last, but not least, I would like to thank my family and my friends for their support, understanding and encouragement during all this time

Nguyen Thu Huong

Trang 5

Contents

LIST OF FIGURES i

LIST OF TABLES ii

LIST OF ABBREVIATIONS iii

PART I INTRODUCTION 1

1.1 Researcher rationale 1

1.1.1 Overview of Arabidopsis thaliana 1

1.1.2 Overview of map-based cloning method 3

1.2 Research’s objective 4

PART II MATERIALS AND METHODS 5

2.1 Materials and equipments 5

2.1.1 Plant material for map-based cloning and growth conditions 5

2.1.2 Equipments 5

2.2 Methods 5

2.2.1 DNA extraction 5

2.2.2 Genetic analysis using SSLP markers 7

2.2.3 Electrophoresis 8

2.2.4 Phenotype analysis 8

2.2.5 DNA purification 9

PART III RESULTS AND DISCUSSION 11

3.1 Morphological phenotypes of AP-44-1 mutant line 11

3.2 Linkage analysis of the mutation (LRI) 13

3.3 Identification of mutant gene 18

PART IV CONCLUSION 23

REFERENCES 24

Appendices 1 27

Appendices 2 28

Trang 6

LIST OF FIGURES

No of

Figure 7 Locations of the SSLP markers in each chromosome 15

Figure 8 Electrophoresis of wild type plant in mapping line (AP-44-1) for

Figure 9 Example of linkage analysis with markers using wild-type plants

Figure 10 A schematic diagram of the positional cloning Final region of

mapping is 163kb and in this region containing 49 candidate genes

22

Figure 11 Result of PCR analysis with 3 set of primers 32460 25

Trang 7

LIST OF TABLES

Table 1 Result of PCR-based analysis using markers in each

Table 2 Result of PCR-based analysis using markers in chromosome 2 19

Trang 8

Ler-0 Landsberg erecta

NaCl Natri chlorua PCR Polymerase Chain Reaction SSLP Simple Sequnce Length Polymophic TAE Tris-acetate-EDTA

Trang 9

PART I INTRODUCTION

1.1 Researcher rationale

1.1.1 Overview of Arabidopsis thaliana

Arabidopsis thaliana has recently become the organism of choice for a wide

range of studies in plant sciences While genome projects have documented the extent

to which all eukaryotic organisms share a common genetic ancestry, research with

Arabidopsis has clarified the important role that analysis of plant genomes can play in

understanding basic principles of biology relevant to a variety of species, including humans Several plants were recognized as model genetic systems, including maize, tomato, pea, rice, barley, petunia, and snapdragon, but research biologists failed to reach a consensus on which species was most suitable for studying processes common

to all plants Twenty years ago, plant biologists began to search for another model organism suitable for detailed analysis using the combined tools of genetics and molecular biology Plants with effective protocols for regeneration in culture (such as petunia and tomato) were logical candidates, particularly for studies involving

Agrobacterium mediated cell transformation, but attention gradually shifted toward Arabidopsis, a small weed in the mustard family that was first chosen as a model

genetic organism by Laibach in Europe and later studied in detail by Re´dei in the

United States (Mienke et al., 1998)

The modern era of Arabidopsis research began in 1987 with theopening of the Third International Arabidopsis Conference at Michigan State University and the subsequent formation of an electronic Arabidopsis newsgroup Many individuals experienced in the analysis of other model organisms soon began to study Arabidopsis

as a promising model for basic research One important outgrowth of thisincreased

enthusiasm for Arabidopsis research was the drafting in 1990 of a vision statement outlining long-term research goals for the Arabidopsis community These included

saturating the genome with mutations, identifying every essential gene, and sequencing the entire genome by the end of the decade The importance of applying

advances with Arabidopsis to other plants and to solving practical problems in

Trang 10

agriculture, industry, and human health was also stressed A further commitment to

Arabidopsis research was made in 1996 with the establishment of the Arabidopsis

Genome Initiative dedicated to coordinating large-scale sequencing efforts This initiative has become a model for multinational cooperation and has already resulted in more than 30 Mb of genomic DNA sequence being deposited in public databases The remainder of the 120-Mb genome is scheduled to be sequenced by the end of 2000

Arabidopsis has therefore progressed in 20 years from an obscure weed to a respected

member of the “Security Council of Model Genetic Organisms” Here is some recent

advances in Arabidopsis research and summarize features that have made this simple angiosperm a model for research in plant biology (Mienke et al., 1998)

Arabidopsis thaliana is an important model plant for identifying genes and

pursing their function (Rensink et al., 2004) Arabidopsis has been used to as an ideal

model for studying the plant biology and genetics There is ample reason to believe

that Arabidopsis will serve as a resource base for breeders of crop plants and as a

model plant that further the knowledge of plant scientists (Hayashi and Nishimura,

2006) Classified in a member of the mustard and cabbage plants, Arabidopsis thaliana

has many advantages for genetics analysis, including a short life cycle, small size of plant, large number of offspring from self-fertilization, ease of out-crossing and a

relatively small genome (Mienke et al., 1998) It has thus become the focus on genome

project for learning the molecular biology and genetics in flowering plants at the molecular level

Proper leaf development is essential for plant growth and development, and leaf morphogenesis is under the control of intricate networks of genetic and environmental cues Leaf development is one of the fundamental processes ensuring robust photoautotrophic growth for higher plants and mechanisms are in place to coordinate

the establishment of leaf polarities (Byrne et al., 2012). The leaf is the major organ involved in light perception and conversion of solar energy into organic carbon In order to adapt to different natural habitats, plants have developed a variety of leaf forms, ranging from simple to compound, with various forms of dissection The fact that numerous factors have been shown to be able to modulate leaf curvature suggests that higher plants utilize complex regulatory schemes to ensure the proper

Trang 11

development of leaves

lamina development and reinforce the notion that leaf epregulating plant organ morphogenesis

1.1.2 Overview of

map-Various strategies for the identification of plant genes have been used

DNA using molecular markers cloning was used to identify the mutated gene in this studmethod of identifying a mutation gene by looking for linkage to markers thatlocation in the genome is known This process

designing the markers

essentially indirect approach:

containing a mutation by successively excluding all other parts of the genome.molecular markers most widely

sequence length polymorphisms (SSLP), cleaved amplified polymorphic sequences (CAPS) and derived CAPS (dCAPS)

Transcription factors, ABS7/MYB101, is

lamina development and reinforce the notion that leaf epidermis plays critical roles in

regulating plant organ morphogenesis (An et al., 2014)

Figure 1 Arabidopsis thaliana -based cloning method

for the identification of plant genes have been used

DNA using molecular markers was very fast and accurate strategy and map

was used to identify the mutated gene in this study method of identifying a mutation gene by looking for linkage to markers thatlocation in the genome is known This process need to making physical map and

Map-based cloning, also called positionalessentially indirect approach: mapping will narrow down the genetic interval

by successively excluding all other parts of the genome.molecular markers most widely used in mapping experiments at present are simple sequence length polymorphisms (SSLP), cleaved amplified polymorphic sequences (CAPS) and derived CAPS (dCAPS) Simple sequence length polymorphism (SSLP)

is able to alter leaf idermis plays critical roles in

for the identification of plant genes have been used Analyzing

was very fast and accurate strategy and map-based

-based cloning is the method of identifying a mutation gene by looking for linkage to markers that physical

need to making physical map and based cloning, also called positional cloning, is an mapping will narrow down the genetic interval

by successively excluding all other parts of the genome The

used in mapping experiments at present are simple sequence length polymorphisms (SSLP), cleaved amplified polymorphic sequences

Simple sequence length polymorphism (SSLP)

Trang 12

markers are designed based on a unique segment of genomic DNA sequence that contains a simple tandem repeat that distinguishes the genotypes In Arabidopsis, SSLPs are largely based on the (GA)n repeats Cleaved amplified polymorphic sequences (CAPS) markers are designed based on restriction fragment length polymorphisms of PCR amplified fragments, when sequence information of one of the haplotypes is unknown As compared to CAPS markers, SSLPs offer the additional advantage that they do not involve the use of restriction endonucleases and thus avoid

the problems associated with partial digestions (Lukowitz et al., 2000)

As a first step of mapping, the mutant plant was out-crossed with other ecotype wild-type plant F2 seeds are planted and performed phenotype and genotype analysis

F2 seeds were collected from self-pollinated F1 plants PCR analysis was carried out using Simple Sequence Length Polymophic (SSLP) markers and DNA which were prepared from the leaves of F2 plants Recombinant samples were separated from PCR analysis and mutation region was narrowed down in an interval of markers In

Arabidopsis, most of cases in mapping have been successful with 3,000 to 4,000 plants

(Jander et al., 2002) Final step of mapping is sequencing analysis of candidate genes

In this study, we used the SSLP marker that has some advantages following: simple, easy to perform, inexpensive, consistent with the conditions of the laboratory to

identify gene involved in gametophyte development of mutant line, named as AP-44-1

To find a mutant gene, mapping population was conducted and analyzed phenotypic analysis of F2 plants Genomic DNA of F2 plants were prepared from leaves of 689 plants to define chromosomal region based on PCR analysis using SSLP markers

which showed polymorphism between Ler-0 and Col-0 (Figure 5)

1.2 Research’s objective

Although leaf rolled inside phenotype were results of ectopically expressed gene,

our work do demonstrate the utilities of gain-of-function genetic approaches in uncovering potential regulators of plant development Mutant gene may be exploited

in the future for generating curly leaf traits when desired Our work demonstrated that enhanced expressions of R2R3-MYB transcription factors, are able to alter leaf lamina development and reinforce the notion that leaf epidermis plays critical roles in regulating plant organ morphogenesis

Trang 13

PART II MATERIALS AND METHODS

2.1 Materials and equipments

2.1.1. Plant material for map-based cloning and growth conditions

Mutant plant with genetic background of Landsberg erecta (Ler-0) was

out-crossed with Columbia (Col-0) wild-type plant In F2 generation, plants were segregated wild-type plants and heterozygote mutant used for mapping

Seeds were sown in soil (soil and vermiculate mixed at ratio of 1:1) and treated at 4oC for 2-3 days in the dark for dormancy breaking After 2-3 days, seeds were transferred to growth room under the condition of 16 hours light at 23oC and 8 hours dark in 21oC After 2 weeks from seed germination seedlings were transplanted

cold-to 50 hole pot

2.1.2 Equipments

Centrifugal machine HM-150IV (Hanil science industrial), molecular imager ChemiDoc XRS (BIO RAD), tissue layer machine (QIAGEN), PCR machine T100 Thermal Cycler (BIO RAD), Vortex-genie 2 (Scientific industries), agarose (Vivantis), pipet, microwave (Sam Sung), pipets (LABMATE), incubate bath (LABTECH), analytical balance (Sartorius), beaker (Japan), …

2.2 Methods

2.2.1 DNA extraction

DNA (Deoxyribonucleic Acid) is a long stringy molecule that can be extracted from any biological material such as living or conserved tissues, cells and virus particles A number of basic procedures are carried out to isolate and purify DNA First the cell is broken open to expose its DNA This is achieved by blending or grinding the cell The next step involves breaking down and emulsifying the fat and proteins that make up the cell's membrane This is achieved by the addition of both salt and detergent solutions Following this, the DNA is separated from the liquid solution

by the addition of an alcohol and centrifugation This provides the purified DNA ready for use in different applications The ability to extract DNA is of primary importance

Trang 14

to studying the genetic It is also essential for carrying out forensic science, sequencing genomes, detecting bacteria and viruses in the environment The extraction of DNA is pivotal to biotechnology It is the starting point for numerous applications, ranging from fundamental research to routine diagnostic and therapeutic decision-making Extraction and purification are also essential to determining the unique characteristics

of DNA, including its size, shape and function

The genomic DNA was extracted from fresh leaves of individual plants using cetyltriethy-ammonium bromide (CTAB) method with some modifications following below steps:

1.The leaf samples were collected in 2ml tube and put a metal bead

2.Freezing leaf samples in liquid nitrogen and grinding using a Tissue Layer machine

3 Immediately, adding 250µL of CTAB buffer (1,4 M NaCl,20mM Na2, EDTA, 100Mm Tris pH 8, 3% w/v CTAB)

4.After vortexing at room temperature for 15 minutes, adding 250 µL of chlroform/IAA (24:1)

5.Mixtures were centrifuged for 12 minutes at 12000 rpm

6.Supernatant was transferred to 1,7ml tube containing 140 µL of isopropyl alcohol and leave at room temperature for 5 minutes

7.To make a pellet, tubes were centrifuged for 7 minutes at 12,000 rpm and then supernatant were discarded

8.To washed pellet, tubes were centrifuged with 700 µL ethanol for 5 minutes

at 12,000 rpm and removed remainder with pipette

9.The pellet was dried in clean bench and DNA were dissolved in 50 µL autoclaved distilled water (ADW)

10 Finally, store DNA solution at -200C

Trang 15

Figure 2 Process of DNA extraction (CTAB method )

2.2.2 Genetic analysis using SSLP markers

To find the locus of mutation, SSLP (Single Sequence Length Polymorphism) markers were designed Firstly, 13 wild-type samples of mapping lines were analyzed using 10 different markers and a chromosome which is contained mutant gene can be found Next, more markers were designed on mutant chromosome and narrowed the mutation locus down by analyzing of recombinant samples PCR was run at 95 for 2 min to denaturation followed by 40 cycles of 94oC for 30s, 55oC for 15s, 72 oC for 15s, and 72 oC for 5 min to final elongation using 15 µL PCR mixture which contained 1

µL of template DNA, 0.375 µL of 10 pmole/µL marker, 1.5 µL of 10X PCR buffer, 0.3

µL of 2.5 mM dNTPs, 0.075 µL of enzyme and 11.375 µL of ADW

Trang 16

Figure 3 Principle of SSLP mapping

2.2.3 Electrophoresis

Gel electrophoresis is the standard lab procedure for separating DNA by size for visualization and purification Electrophoresis uses an electrical field to move the negatively charged DNA toward a positive electrode through an agarose gel matrix The gel matrix allows shorter DNA fragments to migrate more quickly than larger ones Thus, you can accurately determine the length of a DNA segment by running it

on an agarose gel alongside a DNA ladder PCR products were loaded into 4% agarose (Vitantis Inc.USA) gel in 1X TAE buffer and stained with ethidium bromide Run the gel at 100 V and analyzing gel by Chemidoc XRS camera

2.2.4 Phenotype analysis

Plants were grown under condition of 16 hours light, 23oC/21oC of day/night temperatures in a controlled-environment growth room After 3 weeks, we observed the leaf morphology of mutant lines

Trang 17

2.2.5. DNA purification

Applications such as cloning and sequencing DNA frequently require the purification of DNA fragments from agarose gels or amplification reactions DNA purification can help extract ample amounts of genomic or plasmid DNA sample from

a limited source to satisfy the requirements of researches It also reduce the amount

of contaminants that can compromise the results of research and shorten the shelf-life

of precious samples

In this study, DNA was purified from PCR product following below steps:

1 On the electrophoresis gel, the location which contain band of DNA was cut

to collect DNA and then put into 2 ml of tube

2 Add UB solution with volume is three times as much as gel volume

3.The tube is incubated in 60 oC for 10 min and after that add isopropanol (it is the same with the volume of gel)

4.Transfer 800 µL solution into tube containing filter membrane and centrifuge for 1min at 7000 rpm

5.Discard the solution, and then add 750 µL WB (ethanol 75%)

6.Centrifuge for 1min at 13,000rpm and then remove the supernatant, add 750

µL WB (ethanol 75%)

7.Spin for 3 min at 13,000 rpm

8.Transfer tube containing filter membrane into 1.5 mL tube

9.Add 35 µL EB solution and incubate at room temperature 1 min

10 Centrifuge for 1 min at 13,000 rpm

11. Store DNA elution at -20 oC

Trang 18

Figure 4 Process of DNA purification

Figure 5 Procedure of AP-44-1 map-based cloning

Tiêu đề	Map-Based Cloning of Leaf Rolled Inside (LRI) Mutation in Arabidopsis thaliana
Tác giả	Nguyen Thu Huong
Người hướng dẫn	Prof. Park Soon Ki, PhD. Pham Bang Phuong
Trường học	Thai Nguyen University of Agriculture and Forestry
Chuyên ngành	Biotechnology
Thể loại	Thesis
Năm xuất bản	2016
Thành phố	Thai Nguyen

Định dạng
Số trang	36
Dung lượng	0,95 MB