Nghiên cứu sự đa dạng di truyền của nấm Metarhijum anisopline


TR 

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






S 








Metarhizium anisopliae 





GEN Pr1 -ITS



  SINH HC


: 2003 - 2007




: 










9/2007


TR 

*************

KHO















Metarhizium anisopliae 




GEN Pr1 -ITS

: 



:
ThS.  




TS. 







9/2007


iii
N

 


 

 


 TS, ThS. , CN. Lu Ph L 

 





 
, 





 

 












 , 



i








 












.












iv
T


 Metarhizium
anisopliae Pr1 -ITS  

.


 

Metarhizium anisopliae 
. 














 


























Pr1 -ITS

-  Metarhizium anisopliae 










Metarhizium anisopliae var. anisopliae.
- Pr1 10 Metarhizium anisopliae. 




Pr1 10 

. Pr1 7 



1091bp.
-  










 Metarhizium anisopliae trong


















 .
 95-100%,  g





94-99% 





Pr1
-  4  2  9 




. 











1.
- 5.8S v2 






Metarhizium
anisopliae 







 .  99-
100%  92-100% 






ng rDNA-ITS.


v
M

 TRANG


 .............................................................................................................iii
 .................................................................................................................. iv
 .................................................................................................................. v
 ...................................................................................viii
 ............................................................................................... ix
 ................................................................................................ x
1. 



 ................................................................................................................ 1
1.1.  ............................................................................................................. 1
1.2.   .................................................................................... 2
1.2.1.  ...................................................................................................... 2
1.2.2. 

 .............................................................................................. 2
 ........................................................................................... 2
2.  ...................................................................................... 3
2.1. 



Metarhizium anisopliae ............................................................... 3
2.2.  ................. 6
2.3.  ................................................................................................. 7
2.4.  ....................................................
2.5. Metarhizium anisopliae
 ............... 8
2.5.1.  ............................................................................... 8

2.5.2.  ............................................................................... 9
2.6. 



Pr1 .............................................................................................. 10
2.7. 



Pr1 ..................................................................................... 10

vi
2.8.

-rDNA ..................................................................................... 10
2.8.1. 









 ...................................................... 10
2.9. 



















Metarhizium anisopliae 



Pr1 -ITS ................................. 12
2.9.1. 





 ............................................................................. 12
2.9.2. 




 .............................................................................. 14
2.10. 





Pr1 -ITS ....................................... 14
3.  ............................................. 16
3.1.  ......................................................................................... 16
3.1.1.  ................................................................................................... 16
3.1.2.  .................................................................................................... 16
3.2.  ............................................................................................. 16
3.2.1.  ...................................................................................................... 16
3.2.2.  .................................................................................................... 17
3.2.3.  ...................................................................................... 17
3.3.  ........................................................................................... 18
3.4.  .................................................................................... 18
3.4.1.














 Metarhizium
anisopliae ............................................................................................................. 18
3.4.2. Pr1 ................................................................................. 18
3.4.3. 



-ITS ..................................................................... 21
3.5. 









Pr1  .................................... 22
3.6. 





 ................................................................................ 22

3.7. Tinh s..................................................................... 24
3.8. 







...................................................................... 24
4.  ............................................................................... 26
4.4. 





Metarhizium anisopliae 





vii


 ............................................................................................................................. 26
4.5. 






 ...................................................................... 28
4.6. 





1-5.8S-ITS2. .................................................... 28
4.7. 









ITS1-5.8S-ITS2 .................................................... 29
4.8.  Pr1...36
4.9. 








Pr1………………………………………………………36
5.  .................................................................................... 42
5.4.  .......................................................................................................... 42
5.4.2. Pr1 ............................................................. 42
5.4.3. -ITS ................................................ 42
5.5.  ........................................................................................................... 43
5.6.  ........................................................................................... 43
6.  ...................................................................................... 44
7.  ............................................................................................................... 47








viii


 
 
RBCQ9 
BDQ96 n 9 6
BDTV5 
BDTN 
BDBD7 :   7
SCLLLA 
RNBT 

RNLA : 
ng : nano gram
nm : nano mol
g : micro gram
m : micro mol
M 
l : micro 
TE : tris EDTA
dNTP : deoxyribonucleotide  5  trphosphate
TAE : tris acetic EDTA
UI : unit international
bp : base pair
Kb : kilo base
ctv 
PCR : Polymerase Chains Reaction
Rpm : Revolutions per minute











ix
NG

 TRANG
 2.1 



Metarhizium anisopliae ............................................... 3
 3.1  Metarhizium anisopiae 
















 ........................................................................................ 16
 3.2  Pr1 




METPR1/METPR4 ......................................................................................................... 19

 3.3  Pr1 




METPR2/METPR5 ......................................................................................................... 20
 -ITS .... .21
Bng 3.5  ................................................ 23
Bng 4.1  Metarhizium anisopliae 














-ITS. .................................................................................... 30
4.2 












Metarhizium anisopliae trong












A-ITS ..................................................................... 31
Bng 4.3 












Metarhizium anisopliae trong




 











-ITS.................................................33
4.4 Metarhizium anisopliae 






Pr1 ....................................................................................................... 37
4.5 












Metarhizium anisopliae trong










Pr1 .................................................................................. 38
Bng 4.6 












Metarhizium anisopliae trong




 









Pr1..............................................................40


x


 TRANG

Metarrhizium anisopliae 
nhau ........................................................................................................ 5
2.2 






 ............................................................. 12
3.1 









Pr1 .............................. 18
3.2 











 ........................... 21

3.3 











 ................ 24
3.4 









 ..................................... 25
DNA tng Metarrhizium anisopliae ...................................... 26
4.2 










Metarhizium
anisopliae 







 ......................................... 27
4.3 



Metarhizium anisopliae 



 ........... 28
 rDNA-ITS ........................................................ 28
Metarhizium anisopliae c
d -ITS ........................................................ 32





Metarhizium anisopliae 
 gi









-ITS.................................... 34
i gen Pr1 ..................................................................... 36
Metarhizium anisopliae c
d gen Pr1 ...................................................................... 39
4.9 



Metarhizium anisopliae 
 gi







gen Pr1 ................................................. 41

1

1. 

1.1. 
 


 

 
 

  .             a ra
 
   



Trong  
 Metarhizium anisopliae- 
  Metarhizium anisopliae 

      

2

 
 N

         RAPD, RFLP, AFLP, Southern blot,
Sequence v.v.. 

, Bm
s 
Metarhizium anisopliae  Pr1 -
ITS .
1.2.  
1.2.1.












 Metarhizium anisopliae















 












Pr1 -ITS.
1.2.2. 
- 












 Metarhizium anisopliae








.
- Pr1 -



.
-   Pr1 -





















Metarhizium anisopliae.
1.3. 
Metarhizium anisopliae  


, 

3

 2. 
2.1. Metarhizium anisopliae.

:  Metarhizium anisopliae.
 Fungi
 Ascomycota
 Sordariomycetes
 Hypocreles
 Clavicipitaceae
 Metarhizium
 Metarhizium anisopliae




 


 


4

Metarhizium anisopliae  : M. anisopliae var. major 
  14,0 (18,0) M. anisopliae var. anisopliae
  8,0) m. 
Metarrhizium anisopliae) 

 Metarhizium anisopliae          

 

Metarhizium anisopliae 
            
  Metarhizium anisopliae        
Giang, 1997).
  Metarhizium anisopliae        Aedes
aegypti, Anopheles stephensi  Clex pipiens      
Scotinophara coarctata Schistocera gragaria 
Nasutitermes exitiosus 

M. anisopliae 


20
o



         Metarhizium anisopliae  
Nematod (Heterodenas chachatii Globodera rostochensis

    
5



Metarhizium anisopliae.
Metarhizium anisopliae:
- 
-       
o
          

2

2

               Aeromonas 
in vitro).
- 
o


o

-  30
o

o
C trong

- 
o
 8,5.
- Metarhizium anisopliae         











: Metarhizium anisopliae 

6

2.2. .

 

  









g

Aspergillus  
 










7



n.






 

 

 
               
    




2.3. .

  Beauveria bassiana,    : Metarhizium
anisopliae .Trong 
.
2.4. 
 Metarhizium anisopliae  Beauveria       

8


 
  
Beauveria bassiana bi 
- 


B. bassiana 
B. bassiana 


 M. anisopliae          
Nomadacris succincta M. flavoviride 

            
2004).
2.5. Metarrhizium anisopliae

2.5.1. 
Metarhizium
 Metarhizium 

              
Metarhizium anisopliae 

Beauveria bassiana 
 
9

        
Long.


             Beauveria bassiana,
Metarhizium anisopliae  Normurea rileyi
              

Hemiptera.
2.5.2. 

       
ang, 1997).
           

-
        Pandora neoaphidis,
connidiobolus thromboides, C.coronatus, Entomophthora planchoniana  neozygites
fresenii) Beauveria basssiana, Verticillium lecanii).

          Metarrhizium anisopliae, Beauveria
bassiana  Verticillium lecanii 
 
                
Metarhizium anisopliae Beauveria bassiana  
Verticillium lecanii 

10


Metarhizium anisopliae 
                

2.6. Gii thiu gen Pr1
Nhng n 
nhp trc tip qua lp cutin. sn xut nhng enzyme ngong trc
ti   p v bc c   Nm Metarhizium sn xut mt protease
subtilisinlike ngoc gPr1. Pr1 a lp cutin. Pr1

c tng ht tin cht ln (40.3 kDa) cha mu, mt tin
t protein . Cp ca Pr1  ng ln (30-
60%) vi nhng enzyme c  p subtilisin c   

2.7. Pr1
 



           

2.8. -rDNA
2.8.1. 
     


u

11






1996).



 

              
                

             

            
rRNA .






 -LSU-
      
2001).
12




     





2:     rDNA
2.9. 





















 Metarhizium

anisopliae 



Pr1 -ITS
2.9.1. 






Savita Bagga, Gang Hu, Steven E. Screen, Raymond J. St.
















 -




M.anisopliae
4 :
Pr1C

13

N: 

Pr1A, Pr1B, Pr1G, Pr1I, Pr1K.
Pr1D, Pr1E, Pr1F, Pr1J.
Pr1H .
Cheng Wang, Milton A. Typas, Tariq M. Butt (2002) 










Nested-












 Pr1A Pr1B 










 



  .


:  Pr1 , 













.




















. 










 t Pr1 








n .
Sorayac C . M.. 

 (1997) 

 

















Metarhizium 


















 Pr1  






 . 




40 Metarhizium 



15 
4 .


 (2002) 










3


18

5

28














81 28  5.8S  


 Metarhizium anisopliae 

















.
Malena P. Panton, Annoula Mavridou, Milton A.Typas (2003) 





















 -














40 Metarhizium.
N.D.Pipe, D.Chandler, B. W. 

.B.Heale (1995) 








RFLP-








 (rDNA) 






Metarhizium  M.anisopliae var. anisopliae, M.anisopliae var. majus,
M.album M.flavoviride .
14

Marilena Aquino de Muro, Samina Mehta, David Moore (2003) 








,  50 Beauveria
bassiana.  B.bassiana
2%.



 (1998) 









 Metarhizium
anisopliae var. anisopliae 

















. 











 , 25 d 20
.


 (2000) 





































 . 








 10 


.
Curran  (1994) 









 
Metarhizium 









.


 (1994) 

















Metarhizium.
2.9.2. 















 Metarhizium
anisopliae 



 Pr1 -ITS  . 












 

Metarhizium anisopliae 
.
2.10. Pr1 rDNA-ITS

RFL


 D              

Metarhizium anisopliae            
15



              
Metarhizium anisopliae 
 
Pr1 (St Le
Metarhizium anisopliae - 5.8S  ITS2.