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Iti:
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KHOA HC
KHOA KHOA HC S SNG
NG TH HOA

NHN GEN CYSTATIN MT S GING NG T

C KH NNG CHU HN KHC NHAU

NGI HNG DN KHOA HC: TS. NGUYN V THANH THANH

THI NGUYN - 2012

Ti liu chia sNI

ti: wWw.SinhHoc.edu.vn
DUNG
M U

...

2
3

VT LIU V PHNG PHP NGHIN CU

..

KT QU NGHIN CU V THO LUN

..

KT LUN V NGH

Ti liu chia s
ti:
wWw.SinhHoc.edu.vn
1.
M U
1. M U
1.1. L do chn ti
- Ng (Zea mays L.): l mt trong 5 cy ng cc quan trng
cung cp lng thc cho con ngi v vt nui.
- Hn hn nh hng n nng sut cy trng ni chung v
cy ng ni ring.
Chn v m rng din tch trng cc ging ng lai c
nng sut cao, c kh nng chu hn tt
Nghin cu kh nng chu hn ca cy ng
Cc nghin cu u thng nht rng c tnh chu hn
ca cy trng ni chung v cy ng ni ring do nhiu gen
quy nh, trong c gen Cystatin.

Ti liu chia s
1. ti:
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U
- Cystatin c bn lun nhiu bi mi lin quan ca n ti tnh
chng chu yu t bt li ca ngoi cnh nh: nng, lnh,
mn, hn, c bit l hn.

- Xut pht t nhu cu thc tin ca vic chn to ging ng

theo hng nng cao kh nng chu hn, chng ti tin hnh
ti nghin cu: Nhn gen Cystatin mt s ging ng
t c kh nng chu hn khc nhau.

M
U
Ti liu chia s1.ti:
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1.2. Mc tiu nghin cu
nh gi kh nng chu hn ca mt s ging ng giai
on cy non.
nh gi cht lng ht ca mt s ging ng trn
phng din ha sinh.
Nhn gen cystatin mt s ging ng c kh nng chu

hn khc nhau.

M
U
Ti liu chia s1.ti:
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1.3. Ni dung nghin cu
Phn tch mt s c im hnh thi ht ca 5 ging ng
nghin cu
Xc nh mt s ch tiu ha sinh ht ca 5 ging ng nghin
cu
nh gi nhanh kh nng chu hn ca cc ging ng giai
on cy non
Tch chit DNA tng s.
Nhn gen cystatin bng k thut PCR.
To dng gen cystatin hai ging ng: mt ging chu hn tt
v mt ging chu hn km.
Tch plasmid ti t hp hai ging ng to dng.

chia
ti: wWw.SinhHoc.edu.vn
2. Ti
VTliu
LIU
Vs
PHNG
PHP NGHIN CU
2.1. Vt liu
Bng 2.1. Cc ging ng nghin cu
STT

Tn ging

LVN10

LVN99

LVN145

LVN885

C919

2.
VT
LIU
V
PHNG
PHP
NGHIN
CU
Ti liu chia s ti: wWw.SinhHoc.edu.vn
2.2. Phng php nghin cu
1. Phng php sinh l, ha sinh
- nh gi nhanh kh nng chu hn: L Trn Bnh v cs (1998)
- nh lng lipid tng s: Phm Th Trn Chu v cs (1998)
- nh lng protein tan tng s: Lowry
- Xc nh hot enzyme amylase: phng php c l
- Xc nh hot protease: phng php c l
2. Phng php sinh hc phn t
- Phng php tch chit DNA tng s: Gawel v cs (1991)
- nh lng v kim tra tinh sch ca DNA tng s: o hm
lng trn my quang ph v in di
- K thut PCR: theo Mullis v cs (1985)

2.
VT
LIU
V
PHNG
PHP
NGHIN
CU
Ti liu chia s ti: wWw.SinhHoc.edu.vn

Bng 2.3. Thnh phn phn ng PCR nhn gen Cystatin

Thnh phn

STT

Th tch (l)

Nc kh ion

11

PCR Buffer (10X)

2,5

MgCl2 (25 mM)

2,5

DNTPS (25 mM)

Mi xui (10 pM/l)

Mi ngc (10 pM/l)

DNA khun (50 ng/l)

Taq polymerase (5unit/l)

Tng th tch

25

2. Ti
VTliu
LIU
Vs
PHNG
PHP NGHIN CU
chia
ti: wWw.SinhHoc.edu.vn
Chu k nhit
30 chu k

1 chu k

94 0C
3

1 chu k

94 0C
72 0C

30

72 0C
10

52 0C
1

40 C

Ti liu chia s ti: wWw.SinhHoc.edu.vn

2. VT LIU V PHNG PHP NGHIN CU

- Phng php tinh sch sn phm PCR: b kt ca hng Bioneer
- Phng php gn gen vo vector tch dng pBT
Bng 2.4. Thnh phn phn ng gn gen vo vector pBT

STT

Thnh phn

Th tch (l )

Buffer T4 ligase (10 X)

2,0

DNA

7,0

Vector pBT

1,0

T4 ligase (2 unit/ l)

1,0

H2O

9,0

Tng

Hn hp c 220C trong 1,5 gi

20

chia
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2. Ti
VTliu
LIU
Vs
PHNG
PHP NGHIN CU
2.2. Phng php nghin cu
- Bin np vector ti t hp mang gen cystatin vo t bo kh
bin E.coli DH5: sc nhit, 42oC,1 pht
- Phng php kim tra sn phm chn dng: PCR t dch khun
lc
- Tch chit plasmid ti t hp: b kt ca hng Bioneer
3. Phng php x l s liu
- Phng php thng k bng chng trnh Excel

Tm tt quy trnh tch dng gen Cystatin

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Sn phm PCR

Gn vo vertor pBT

Bin np vo E.coli DH5

Nui cy trn LB c

(Pepton, cao nm men, NaCl, agar)

Chn khun lc trng

Nui trong LB lng

PCR

Tch plasmid ti t hp

(Pepton, cao nm men, NaCl)

Ti
liu
chiaNGHIN
s ti: wWw.SinhHoc.edu.vn
3.
KT
QU
CU V THO LUN
3.1. c im hnh thi, ha sinh ht ng
3.1.1. c im hnh thi v khi lng 100 ht ca cc ging ng
nghin cu
Bng 3.1. c im hnh thi, khi lng ht ca 5 ging ng nghin cu
Mu sc v

KL ca 100 ht

ht

(gam)

Bn

Vng cam

26,86 0,01

LVN99

Bn

Vng cam

21,45 0,45

LVN145

Bn rng nga

Vng cam

25,91 0,52

LVN885

Bn

Vng cam

22,83 0,11

C919

Bn rng nga

Vng cam

25,43 0,27

STT

Tn ging

Hnh dng ht

LVN10

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3. KT QU NGHIN CU V THO LUN

3.1.2. Hm lng protein, lipid trong ht ng
Bng 3.2. Hm lng protein, lipid trong ht ca 5 ging ng
nghin cu
STT

Tn ging

Hm lng protein

Hm lng lipid

(%)

(%)

LVN10

10,27 0,01

4,67 0,01

LVN99

12,27 0,01

4,67 0,01

LVN145

8,80 0,01

3,33 0,01

LVN885

10,27 0,006

3,00 0,01

C919

10,93 0,01

4,33 0,02

3.
KT
CU V THO LUN
Ti
liuQU
chiaNGHIN
s ti: wWw.SinhHoc.edu.vn
3.1.3. Hot ca enzyme - amylase
Bng 3.3. Hot ca - amylase
Tn mu

Hot amylase
(D d, mm)

LVN10

13 0,25

LVN99

12 0,17

LVN145

13 0,14

LVN885

11 0,15

C919

18 0,12

Hnh 3.1. Hot - amylase

1.LVN885 2.LVN10 3.C919
4.LVN145 5.LVN99 6.H2O

3.
KT
CU V THO LUN
Ti
liuQU
chiaNGHIN
s ti: wWw.SinhHoc.edu.vn
3.1.4. Hot protease ca 5 ging ng nghin cu
Bng 3.4. Hot protease
Tn mu

Hot protease
(D d, mm)

LVN10

14 0,21

LVN99

16 0,12

LVN145

17 0,15

LVN885

12 0,12

C919

17 0,11

Hnh 3.2. Hot protease

ca 5 ging ng nghin cu
1.C919; 2.LVN10; 3.LVN99
4.LVN145; 5.LVN885; 6.H2O

3.
KT
CU V THO LUN
Ti
liuQU
chiaNGHIN
s ti: wWw.SinhHoc.edu.vn
3.1.5. Chiu di r ca cc ging ng giai on cy
non sau hn
Bng 3.5. Chiu di r ca 5 ging ng sau hn

STT

Tn ging

Kch thc (cm)

LVN10

25,33 0,35

LVN99

26,80 0,19

LVN145

28,70 0,17

LVN885

18,73 0,23

C919

30,80 0,11

3.2.
Kt
qu
nh
gi kh
nng chu hn giai
Ti
liu
chia
s ti:
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on cy non
Kh nng chu hn ca 5 ging ng giai on cy non c
xp theo th t gim dn th hin trong bng 3.6.
Bng 3.6. Kh nng chu hn giai on cy non ca 5 ging ng

STT Tn ging

Ch s chu hn

C919

12920

LVN10

10110

LVN145

8890

LVN99

7680

LVN885

2300

3.2.
nh
gi wWw.SinhHoc.edu.vn
kh nng chu hn
TiKt
liuqu
chia
s ti:
giai on cy non

%CPH9

%CKH9

%CKH3
1.000
0.900
0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000

%CPH3

LVN10
LVN99
%CKH5

LVN145
LVN885

C919

%CPH7

%CPH5

%CKH7

Hnh 3.3. th rada biu din kh nng chu hn ca 5 ging ng

Ti
liu
chia
s
ti:
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3.3. Kt qu tch dng gen Cystatin
ng
3.3.1 Kt qu tch chit DNA tng s t l ng non

Hnh 3.5. Hnh nh in di DNA tng s ca 5 ging ng nghin cu

1.LVN10; 2.LVN99; 3.C919; 4.LVN885; 5.LVN145

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3.3.1. Kt qu tch chit DNA tng s t
l ng non
Bng 3.7. Ph hp ph DNA bc sng 260 nm v
280 nm ca 5 ging ng nghin cu
Tn

A260

A280

A260/A280

ging

Hm lng
DNA(ng/l )

LVN10

0,317

0,164

1,933

792,50

LVN99

0,260

0,135

1,930

650,00

LVN145

0,243

0,130

1,870

607,50

LVN885

0,260

0,140

1,857

650,00

C919

0,300

0,158

1,899

750,00

Ti
liu
chia
s
ti:
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3.3.2. Kt qu nhn gen Cystatin
cy ng

500bp
250bp

Hnh 3.6. Hnh nh in di kt qu PCR nhn gen Cystatin 2 ging ng

1.LVN885; 2.C919; M.Marker 1kb

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chia
s
ti:
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3.3.3. Kt qu tinh sch sn phm
PCR
1

500bp
250bp

Hnh 3.7. Hnh nh in di sn phm thi gel ca 2 ging ng

1.LVN885; 2.C919; M.Marker 1kb

Ti liu
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3.3.4.
Kt chia
qu tch
dng
gen Cystatin cy ng

Hnh 3.8. Hnh nh khun lc t bo E.coli DH5 cha

vector ti t hp mang gen Cystatin

Ti
liu
s ti:
3.3.5.
Ktchia
qu chn
lcwWw.SinhHoc.edu.vn
plasmid ti t hp bng
conoly-PCR
1

500bp
250bp

Hnh 3.9. Hnh nh in di sn phm colony PCR ca 2 ging ng

1.LVN885; 2.C919; M.Marker 1kb

Ti3.3.6.
liu Kt
chiaqu
s ti:
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tch
plasmid ti t hp
1

Hnh 3.10. Hnh nh in di tch plasmid ti t hp

mang gen Cystatin 2 ging ng
1. Plasmid mang gen Cystatin ging ng LVN885
2. Plasmid mang gen Cystatin ging ng C919

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ti: wWw.SinhHoc.edu.vn
4. chia
KTs
LUN
V NGH
Kt lun
- Khi lng 100 ht ca 5 ging ng nghin cu dao ng trong
khong 21,45 g (LVN99) n 26,85 g (LVN10), chiu di r giai
on cy non sau 9 ngy gy hn c khch thc dao ng t 18,73
cm (LVN885) 30,80 cm (C919).
- Cc ging ng nghin cu c hm lng protein dao ng trong
khong 8,80% (LVN99) - 12,27% (LVN145). Hm lng lipid dao
ng t ng trong khong 3,00% (LVN10) - 4,67% (LVN99 v
LVN10). Ging C919 c hot enzyme amylase mnh nht (18
mm) v ging LVN885 c hot amylase yu nht (11 mm).
- Kh nng chu hn ca cc ging ng c s khc bit: Ging C919 l
ging chu hn tt nht, ging LVN885 l ging chu hn km nht.
- nhn bn, chn dng thnh cng gen Cystatin mt ging ng
chu hn tt (C919) v mt ging ng chu hn km (LVN885).

KT
V NGH
Ti liu 4.
chia
sLUN
ti: wWw.SinhHoc.edu.vn
ngh
c trnh t gen cystatin 2 ging ng to dng trn
phc v vic xc nh ch th phn t v chuyn gen nhm to
cy ng c kh nng chng chu hn tt.

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