Khai thác dữ liệu ESTs ở chi cam chanh cho việc phát triển marker phân tử SSR

i
BỘ GIÁO DỤC VÀ ĐÀO TẠO
TRƢỜNG ĐẠI HỌC NÔNG LÂM TP. HỒ CHÍ MINH
BỘ MÔN CÔNG NGHỆ SINH HỌC
************








KHÓA LUẬN TỐT NGHIỆP
KHAI THÁC DỮ LIỆU ESTs (EXPRESSED SEQUENCE
TAGs) Ở CHI CAM CHANH (CITRUS) CHO VIỆC PHÁT
TRIỂN MARKER PHÂN TỬ SSR (SIMPLE SEQUENCE
REPEATS)


Ngành học: CÔNG NGHỆ SINH HỌC
Niên khóa: 2003-2007
Sinh viên thực hiện: LƢU TRẦN CÔNG HUY





Thnh ph H Ch Minh
Thng 9/2007

ii
LỜI CẢM ƠN













  




m 2007






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TÓM TẮT KHOÁ LUẬN
LƢU TRẦN CÔNG HUY, Đại Học Nông Lâm TP. Hồ Chí Minh, tháng
07/2007. “KHAI THÁC DỮ LIỆU ESTs (EXPRESSED SEQUENCE TAGs) Ở
CHI CAM CHANH (CITRUS) CHO VIỆC PHÁT TRIỂN MARKER PHÂN
TỬ SSR (SIMPLE SEQUENCE REPEATS)”



 



                
xpressed Sequence Tags), trong
citrus)
Simple Sequence Repeats




sau:



3

iv
4
(Citrus), 


5
Kết quả
Citrus 
C
               

 
BUILDING
SSRs DATABASE of Citrus. 
       

-
. -
  -  
Tristeza -value = 10
-10

v
ABSTRACT
LUU TRAN CONG HUY, NONG LAM UNIVERSITY, DATA MINING
FOR DEVELOPING SIMPLE SEQUENCE REPEATS (SSR) MARKER IN
EXPRESSED SEQUENCE TAGS (ESTs) FROM CITRUS
Supervisor:


The research was carried out at the department of biotechnology at Nong
Lam University.
Recent advances in genomic technologies have generated a vast amount of
publicly available expressed sequence tags (ESTs) in Citrus. These data can be
mined to identify Simple sequence repeats (SSRs) or microsatellites. These SSRs
are useful because of a broad range of application, such as genome mapping and
characterization, phenotype mapping, marker assisted selection of plant breeding,
additional map-based cloning of important genes. Moreover, this method of
developing SSR marker from ESTs is inexpensive comparing to the traditional
methods.
Methodology
1) We used perl script to receive EST sequences from database NCBI
2) Finded and separated SSRs include in ESTs database
3) We were learning about relationship database model to used to saved

nucleotide, SSRs citrus sequences data and created database contain them.
4) Finding SSR which are homologous with tristeza virus resistance gene.
5) Designed web that contain database control software to share information
with users
Results:
28,241 SSR-containing ESTs (EST-SSRs) were identified by analyzing
191,110 ESTs sequences belonging to Citrus in dbEST division of GenBank.
19,755 primers, which were filtered with repetition checking and BLAST checking,

vi
were designed in flanking regions of SSRs. These data were put into relational
database and integrated SSR finder tool into the BUILDING SSRs DATABASE of
Citrus Website. After cleaning, masking repeat, vector and organelle sequences, the
EST-SSR sequences and the related EST sequences without SSRs were assembled
into contigs and singletons, to reduce redundancy, to enlarge EST-SSRs for primer
designed and to develop consensus sequences. As a result, more 1071 primers were
design for these enlarged EST-SSRs. Using a stringent BLAST search with a
threshold e-value = 10
-10
against typical pathogen resistance gene database in
Citrus, we identified 33 EST-SSRs which are homologous with tristeza virus
resistance gene.

vii
Mục Lục
 .................................................................................................... iii
 ................................................................................. iv
ABSTRACT ...................................................................................................... vi
 ................................................................ xi

Chƣơng 1 ............................................................................................................ 1
 ............................................................................................................. 1
1.1 Đặt vấn đề
1.2.Mục tiêu của khóa luận

Chƣơng 2 ............................................................................................................ 3
 ................................................................................... 3
 ........................................................................... 3
 ........................................................................................... 3

m .................................................................................................... 4
2.1.3  .................................... 6
2.2 EST ............................................................................................................... 7
 .......................................................................................... 7
 ................................................................................... 7
 ............................................. 8
 ............................................... 8
 ......................................................................................... 9
 .................................................................................................. 9
 ............................................................. 10
 ........................................................ 11
 ............................................................................. 12
 ............................................................. 12

viii
2.3.5  ......................................................................... 13
..................................... 15
 ........................................... 16
 ..................................................................................................... 18
 ................................................................................. 18
2.7.1 NCBI ........................................................................................................ 19
 .................................................................................... 19
 .......................................................... 19

Chƣơng 3 ......................................................................................................... 20
 .................................................................... 20
............................. 20
 ............................................................................................ 20
 ......................................................... 20
3.1.2.1 Chương trình Perl ssrfinder_1 .................................................. 20

 BLAST .................. 22
 ..................................... 23
3.1.2.4 Egassembler .......................................................................................... 23
3.1.3 Apache web Server .................................................................................. 24
 ......................................................................... 25

Chƣơng 4 .......................................................................................................... 37
 .......................................................................... 37
4.1  ....................................... 37
EGassembler 
........................................................................................................................... 38
 ..................................................................................... 38
4.2.2  ........................... 39
 .................................... 39

ix
4.3 Assembling .................................................................................................. 41
  .............................. 42
4.4.1 BLASTn: ................................................................................................. 43
4.5. ......................................................................... 45
4.6 tBLASTx ..................................................................................................... 48
4
 ............................................................................................ 49
        
 .......................................................................................... 49
 ...................................................................... 49
SRs (SSRs PAGE) ................................................. 50

Chƣơng5 ........................................................................................................... 52
 ............................................................................... 52

 ...................................................................................................... 52
 ....................................................................................................... 53

 ................................................................................. 54

 ............................................................................................................. 57















x

DANH SÁCH CÁC TỪ VIẾT TẮT


BLAST Basic Local Alignment Search Tool
CGI Common Gateway Interface
CSDL 
DBD Database Driver

DBI Database Interface
DNA deoxyribonucleic acid
EST Expressed Sequence Tag
HTML Hypertext Markup Language
HTTP Hypertext Transfer Protocol
NCBI the National Center for Biotechnology Information
NIG the National Institute of Genetics
NIH the National Institutes of Health
NLM the Nation Library of Medicine
Perl Practical Extraction and Report Language
PHP Hypertext Preprocessior
RDBMS Relational Database Management System
SNP Single Nucleotide Polymorphism
SSCP Single- Strand Conformation Polymorphism
SSR Simple Sequence Repeats
STS Sequence Tagged Site









xi
DANH SÁCH CÁC BẢNG

 .................. 26
  ............................ 26

 ............................................................................. 34
 ....................................................... 34
 ................................................................................ 34
 ....................... 37
 ....................................................... 38
....................................................... 39
....................................................... 39
 ................ 41
 .................................................. 42
 ..................... 43
  ............................................................. 45
 ............................................ 45
.................... 48
 ..................................... 50
 ................................................ 51











xii

DANH SÁCH CÁC HÌNH
 ..................................................................... 6

 ............................................................................ 8
 ................................................. 12
 ............................................. 13
 .......................... 16
 ...... 19

www.NCBI.nlm.nih.gov/genomes/plant/plantlist.html#est) ............................ 27
2 Egassembler .............................................. 29
 .................................................... 30
 ............................................... 31
 ........................................... 31
 ......................................... 32
         -
india.org/ssr/ssr.htm) ......................................................................................... 36
 .......................................... 37
 ................... 40
 ............................................... 41
 ... 42-43
 ............ 44
 .................... 46
 .............. 47
 .............................................................. 49
 ...................................................................... 49
 ........................................................ 50

 ...................................................................................... 51
Chƣơng 1
MỞ ĐẦU
1.1 Đặt vấn đề
   

 
. SSR  
 
T             
marker  
      .         
Microsatellite.







 

1.2.Mục tiêu của khóa luận

  

2
    KHAI THÁC DỮ LIỆU ESTs (EXPRESSED
SEQUENCE TAGs) Ở CHI CAM CHANH (CITRUS) CHO VIỆC PHÁT
TRIỂN MARKER PHÂN TỬ SSR (SIMPLE SEQUENCE REPEATS)
u:
1.          

2. 
 
 

3. 

4. K-
Egassembler)
5. 

6. 

7. 



3
Chƣơng 2
TỔNG QUAN TÀI LIỆU
2.1 Giới thiệu về chi cam chanh
Chi Cam chanh (Citrus)  trong 
(Rutaceae).
  -15 m

 -
 

4--
 


2.1.1 Vị trí phân lọai
 Plantae
 Magnoliophyta

 Magnoliopsida
 Rosidae
 Sapindales
 Rutaceae
Chi Citrus


4
2.1.2 Đặc điểm

terpen 
 
vitamin C flavonoit 
                

Citrus 
 
,
, , chanh ta, ,  
FortunellaPoncirus, Microcitrus,
Eremocitrustrong chi Citrus.

Citrus sinensis x Poncirus trifoliata Citrus aurantium



5
Citrus Unshiu

Citrus x paradisi






Citrus Sinensis















Citrus Clementina
6

2.1.3 Sâu hại và bệnh tật
Bệnh do virus
Virus citrus 

 


             
vir


Virus Tristeza (CTV)
Tristeza 

Tristeza 
Virus Tristeza   










 


Hình 2.1. CTV dƣới KHV điện tử

7

(stem pittin       


   Tristeza    Toxoptera
citricida Kirkaldy. 
     

 


.

2.2 EST
2.3.1 Sơ lƣợc về EST





2.3.2 Nguồn gốc của EST


 
 

base.



8














Hình 2.2: Nguồn gốc của EST
2.3.Sơ lƣợc về phƣơng pháp Microsatellite (SSR)
2.3.1Những khái niệm về kỹ thuật microsatellite
Micro         
(q.v.). M

 

 





microsatellite                

9
microsatelli 


 



 
tandem repeats, Edward; 1991) hay VNTR (variable number of tandem repeats).
- 
- 

             
codominant-

4
- 5.10
-6



 
2.3.2 Giới thiệu chung
2.3.2.1 Tính chất




di-, tri-

nucleotide CA  



10



    

- s


 




              


2.3.2.2 Khuếch đại của microsatellites


                  

 




              



11
2.3.2.3 Những giới hạn của microsatellite
     
             
         


 
               
les


 
c




s 
 



                







12
  
 





2.3.3 Các loại microsatellite
-
Dinucleotide SSR (GT)6
GTGTGTGTGTGT
Trinucleotide SSR (CTG)4
CTGCTGCTGCTG
Tetranucleotide SSR (ACTC)4
ACTCACTCACTCACTC
           
., 1996).
2.3.4 Cơ chế hình thành microsatellite



Quá trình bắt chéo lỗi trong quá trình giảm phân (unequal crossing- over
during meiosis)
.

Hình 2.3 
13

 





Hình 2.4 
2.3.5 Vai trò của microsatellite

 


            
