Xác định Gene liên quan đến tính kháng Virus PMWaV gây bệnh héo đỏ đầu lá trên cây dứa Cayenne bằng phương pháp PCR thoái hóa

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


XÁC ĐỊNH GENE LIÊN QUAN ĐẾN TÍNH KHÁNG VIRUS
PMWaV - Pineapple mealybug wilt associated virus - GÂY BỆNH
HÉO ĐỎ ĐẦU LÁ TRÊN CÂY DỨA CAYENNE BẰNG
PHƢƠNG PHÁP PCR THOÁI HOÁ (degenerated PCR)



Ngành: Công nghệ sinh học
Niên khoá: 2003 - 2007
Sinh viên thực hiện: LÊ MINH THÔNG





09/2007

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


XÁC ĐỊNH GENE LIÊN QUAN ĐẾN TÍNH KHÁNG VIRUS
PMWaV - Pineapple mealybug wilt associated virus - GÂY BỆNH
HÉO ĐỎ ĐẦU LÁ TRÊN CÂY DỨA CAYENNE BẰNG
PHƢƠNG PHÁP PCR THOÁI HOÁ (degenerated PCR)




Giáo viên hƣớng dẫn: Sinh viên thực hiện:
TS. Trần Thị Dung Lê Minh Thông
CN. Lƣu Phúc Lợi




09/2007

iii
LỜI CẢM TẠ







          







, 










 
 
    
             
HCM t




 

 


, .





iv
TÓM TẮT

Ch Minh. Th 9/2007.
 -
Pineapple mealybug wilt associated virus -  


 


             



  




PMWaV - Pineapple mealybug wilt associated virus  




Kết quả:





v
MỤC LỤC



 .......................................................................................................... iii
 ............................................................................................................... iv
 ................................................................................................................ v
 .................................................................. viii
 ...................................................................................... x
 ................................................................................... xii
 .............................................................................. xii
 .................................................................................................................. 1
 ......................................................................................................... 1
 ............................................................................................................ 3
 ............................................................................................................ 3
 .............................................................................................................. 3
 ........................................................................................ 4
 ....................................................................................... 4

.................................................................................. 4
 .................................................................................... 4
Cayenne ........................................................................................... 5
 ....................... 7
 ....................................................................... 8
 ....................................................................... 8
 .................................................................................................... 9
 ................................................................................. 10
 ............................................................................. 10
 ............................................................................................. 11
 .......... 11
 ................................................... 13

vi
 ................................................ 13
 ............................... 14
 ................................................. 16
 ..................................................... 18
- for - gene concept) .................................. 19
 .............................................................. 20
 ............... 24
 ...................................... 24
 ......................... 25
 ............................................................................. 27
 ......................................................... 31
t .................................................................................... 31
 ............................................................................................. 32
 ........................................... 32
 .............................................................. 32
 ........................... 35

 ............................................................ 36
riction Fragment Length Polymorphism) .............. 37
 ..................................................................................... 37
 ............................................................ 37
 ............................................... 39
 ................................................... 39
 ....................................................................................................... 39
  ................................................................ 39
 ............................................................................................................ 39
 ..................................................................................... 39
 .................... 40
 ................................................................................. 41
 ................... 41

vii
  .............. 44
 .. 49
 ............................................................................... 50
 ...................... 50
 ........................................................................................................ 50
 ............................................................................................ 51
4.1. ................................................................................................ 54
 .................. 56
 ...................................................................... 56
 .......................................................... 56
 ............................................................. 57
 ............................................................................ 64
4.2.5. PC .................................................................. 65
 ....................... 66
 ............ 68

 .................................................................................... 69
 ........................................................................................................... 69
 ............................................................................................................ 69
 ...................................................................................... 70



viii
DANH SÁCH CÁC CHỮ VIẾT TẮT

bp base pairs
cs. 
CC coiled coil
cDNA complementary deoxynucleic acid
CTAB cetyl trimethyl ammonium bromide
DNA deoxyribo nucleic acid
dNTP deoxyribonucleotide triphosphate
EB extraction buffer
EDTA ethylene diamine tetra acetic acid
ELISA enzyme-linked immunosorbent assay
EST expressed sequence tag
GLRaV-3 grape leafroll associated virus
KIN kinase
LRR leucine rich repeat
MAS marker assisted selection
MWP mealybug wilt of pineapple
NBS nucleotide binding site
OD optical density
PCR polymerase chain reaction
PCV pineapple closterovirus

PMWaV pineapple mealybug- wilt associated virus
QLT quatity loci trait
RFLP restriction fragment length polymorphism
RGA resistance gene analogs
RT reverse transcription
RT-PCR reverse transcription-polymerase chain reaction
TAE tris  acetate  EDTA
TBIA tissue blot immunoassay
TE tris  EDTA

ix
TIR toll interleukin receptor
TMV tobacco mosaic virus
Tp. HCM 

x
DANH SÁCH CÁC HÌNH

 .................................................................................................... 5
 ..................................................................................... 6
 .................................................. 8
9
 ........................................................... 10
 .......................................................... 11
 ......................................... 20
 ... 22
c NBS-LRR. .............. 23
 ................................. 24

 ................................................................................................................ 27


 ......................................................................................................................... 29
 .............................................................. 34
 ................................................................................... 42
 .............................................................................. 43
 ......................................... 51
 ......................................................... 52
 .............................................................. 53
 ................................................... 53
 ......................... 54
 ........................................................ 55
 ............................ 56
 ........................................................................ 57

 .......................................................................................................................... 58

xi
 .................................................... 59
 ...................................................... 60

2+
....................................................... 61
 ..................................... 62
 ............................................................ 62
 .............................................................. 63
 ....... 65
 .... 66
 ................................. 67
Hind III  ............... 68

xii
DANH SÁCH CÁC BẢNG
 ....................................................................... 22
 ................................... 46
 . 47
 . 47
 ......................................................... 49
 ..................................................................................... 50
  .................................................................. 55
 ... 58



DANH SÁCH CÁC BIỂU ĐỒ
 .................................................. 45






1

Chƣơng 1
MỞ ĐẦU
1.1. Đặt vấn đề
           
 

   






          Pineapple mealybug wilt –
associated virus


 



         
  
 





2

 xem
 






 



 
         




   




        
              




3

1.2. Mục tiêu
 
 

     
 


1.3. Nội dung




1.4. Yêu cầu



 






4

Chƣơng 2
TỔNG QUAN TÀI LIỆU
2.1. Tổng quan về cây dứa
2.1.1. Nguồn gốc và phân loại
     


 



Ananas comosus 




Bromeliaceae
Ananas
Ananas comosus
2.1.2. Đặc điểm thực vật học



    
        

.


5


Hình 2.1. Cây dứa





 30
o

2000 mm 


o
- 1500 mm

 
- 

 
2.1.3. Cây dứa Cayenne


a).

6


Hình 2.2. Cây dứa Cayenne
iV
             
Cayenn- 



              

cam.


 4 







- 
7



2.1.4. Tình hình sản xuất và tiêu thụ dứa trên thế giới và Việt Nam
2.1.4.1. Thế giới
u
- 
 -
   -        
           
            

- 2000


       
             
             


2.1.4.2. Trong nƣớc




  
 T- -  



8

 ,
Tp. 
2.2. Bệnh héo đỏ đầu lá trên cây dứa








Hình 2.3. Ruộng dứa bị nhiễm bệnh héo đỏ đầu lá
2.2.1. Tình hình dịch bệnh và tác hại
Hi
               


              

 

9

               

- 


2.2.2. Triệu chứng








Hình 2.4. Cây dứa và quả dứa Cayenne bị nhiễm bệnh đỏ đầu lá do virus
PMWaV.

 
 

 
 
10

                 

2.2.3. Nguyên nhân gây bệnh


s PMWaV - - 2. 

    Closterovirus Ampelovirus Vinivirus 
- - 
%.
2.2.4. Tác nhân lây truyền bệnh
PMWaV - - Dysmicoccus
brevipes D. neobrevipes 
-  
                





(a) (b)
Hình 2.5. Hai loại rệp lây truyền PMWaV.
Dysmicoccus neobrevipes Beards),
(b) Dysmicoccus brevipes Cockerell).

11


    
u xung


 
 

Hình 2.6. Sự cộng sinh giữa kiến và rệp sáp
2.2.5. Cách phòng trừ






 cho 

2.2.6. Một số nghiên cứu về bệnh héo đỏ đầu lá trên thế giới và Việt Nam
2.2.6.1. Thế giới


closterovirus 
Pineapple closterovirus (PCV).
12

 -body  llman

 


Anti-  
 

                
mang virus.
Dysmicoccus
brevipes  Dysmicoccus neobrevipes         

- 1
-  

  
      -        





2.2.6.2. Việt Nam



  
 
 
13

- 
 


   

  










2.3. Tổng quan vế tính kháng bệnh trên thực vật






                 


2.3.1. Khái niệm về tính kháng bệnh trên thực vật

