TRNG I HC KHOA HC

KHOA KHOA HC S SNG

M HUY HONG

PHN LP GEN DREB1 MT S GING U TNG

KHA LUN TT NGHIP I HC Chuyn ngnh: Di truyn hc

Ngi hng dn khoa hc: TS. Nguyn V Thanh Thanh Thi Nguyn-2011

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

1. M U 2. VT LIU V PHNG PHAP 3. KT QU V THO LUN

4. KT LUN V NGHI

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

1. L do chn ti
- u tng (Glycine max (L.) Merrill ) l cy trng h u (Fabaceae)

- Cy u tng l cy c gi tr kinh t cao

- Kh hn l yu t chnh lm gim nng sut u tng di iu kin canh tc nc tri v s kin thiu nc ngy cng nghim trng. Kh hn c th lm gim 70% nng sut cy trng ni chung - Protein DREB1 l nhn t phin m kch hot cc gen lin quan n tnh chu hn ca thc vt ni chung v ca cy u tng ni ring - T l do trn chng ti tin hnh nghin cu ti: Phn lp gen DREB1 mt s ging u tng nhm to c s cho cc nghin cu tip theo

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2. Mc tiu nghin cu

Phn lp v xc nh trnh t gen DREB1 mt s ging u tng c kh nng chu hn khc nhau

Ti liu chia s dung nghin cu 3. Ni ti: wWw.SinhHoc.edu.vn

- Tch chit DNA tng s ging u tng Cuc vng Yn Sn v ging Vng Ngn sn

- Nhn gen DREB1 ging u tng Cuc vng Yn Sn v

ging Vng Ngn sn bng k thut PCR - Phn lp gen DREB1 ging u tng Cuc vng Yn Sn v ging Vng Ngn sn - Tch plasmid ti t hp mang gen DREB1 ging u tng

Cuc vng Yn Sn v ging Vng Ngn sn

- Xc nh trnh t gen DREB1 ging u tng Cuc vng Yn Sn

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

2.1. Vt liu
Chung ti s dung 2 ging u tng a phng c cht lng tt do Vin Ng an Phng H Ni cung cp

CYS

VNS

Hnh 2.1. Ht ca 2 ging u tng nghin cu

2.2. Ha cht v thit b 2.3. a im nghin cu 2.4. Phng php nghin cu 2.4.1. Phng php tch chit DNA tng s

Phng php tch DNA tng s theo Gawel v cs

nm 1991 c ci tin

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2.4.2. nh lng v kim tra tinh sch ca DNA tng s Kim tra tinh sch ca DNA bng 2 phng php (1) Phng php in di

(2) Phng php quang ph hp thu

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2.4.3. K thut PCR
Nhn gen bng k thut PCR theo Mullis v cs nm 1985. Cp mi nhn gen DREB1 c thit k da trn c

s ca 2 trnh t mang ma s AY802779 v FJ965342 trn

Ngn hng gen quc t (NCBI) Bng 2.3. Cp mi nhn gen DREB1
Mi DREB1soyF DREB1soyR Trinh t mi 5 GGATCCATGTTTACCTTGAATC 3 3 GAATTCTTAAATTGAGAAATTCCA5 Nhit gn mi 51.00C 51.00C

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Bng 2.4. Thnh phn phn ng nhn gen DREB1
STT Thnh phn phn ng PCR Th tch (l)

1
2 3

Buffer PCR (10X)

dNTP (5 mM) Mi xui (10 pmoles/l)

2,5
2,5 1

4
5 6 7 8

Mi ngc (10 pmoles/l)

Taq polymerase (1/l ) DNA khun (50 ng/l) MgCl2 (25mM) Nc Tng

1
0,25 3 2,5 12,25 25

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Bng 2.5. Chu k nhit cho phn ng PCR
Bc Phn ng Nhit (0C) Thi gian Chu k 1

1 2 3 4 5 6

Bin tnh Bin tnh Gn mi Ko di chui Hon tt ko di Kt thuc phn ng

94 94 51 72 72 4

3 pht 1pht 1pht 1 pht 30 giy 10 pht

30 1

2.4.4. Phng php tinh sch sn phm PCR Sn phm c tinh sch (thi gel) theo b Kit AccuPrep PCR Purification (Bioneer-Hn Quc) 2.4.5. To vector ti t hp Bng 2.6. Thnh phn phn ng gn gen vo vector
STT 1 2 3 4 Thnh phn phn ng T4 Ligase Buffer T4 DNA Ligase pTZ57R/T Vector PCR product Th tch (l) 1 1 1 5

Nc ct kh ion
Tng

2
10

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2.4.6. Bin np v nui cy t bo mang vector ti t hp Bin np sn phm kt ni trn vo t bo kh bin E.coli

chng DH5 theo phng php sc nhit

Ti liu s c mt ca on gen gn vo plasmid 2.4.7 Kim trachia s ti: wWw.SinhHoc.edu.vn

S dung enzym ct gii hn BamHI v XbaI kim tra s c mt ca gen DREB1 trong plasmid ti t hp Bng 2.7. Thnh phn phn ng ct plasmid ti t hp
STT Thnh phn phn ng Th tch (l)

1
2

Buffer Tango 10X

BamHI enzym

1
0,5

3
4 5

XbaI enzym
DNA plasmid Nc ct kh ion Tng

0,5
3 5 10

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2.4.8. Gii trinh t v x ly s liu
- Thc hin phn ng PCR gn mi hunh quang
- Phn tch, x l chui gen v s liu - Tinh sch sn phm phn ng gii trnh trnh t

KT QU V THO LUN
3.1. Kt qu tch chit DNA tng s
1 1 2 2

Hinh 3.1. DNA tng s ca 2 ging u tng CYS v VNS. Ky hiu: 1: CYS; 2: VNS

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Bng 3.1. Ph hp thu DNA bc sng 260nm v 280nm ca hai ging u tng CYS v VNS

Tn ging A260
CYS
VNS

A280
0,170
0,128

A260/A280
1,911
1,813

Hm lng DNA (ng/l)

662
572,5

0,325
0,232

3.2. Kt qu nhn gen DREB1

M 1 1 2 2

750bp 500bp 250bp

717bp

Hinh 3.2. Kt qu in di sn phm PCR nhn gen DREB1 ca 2 ging u tng CYS v VNS. Ky hiu: M: Marker; 1: CYS; 2: VNS

3.3. Kt qu tch dong gen DREB1

M 1 2

2800 bp 717 bp

Hinh 3.5. Kt qu in di sn phm ct plasmid ti t hp bng BamHI v XbaI M: Marker 1kb; 1: CYS; 2: VNS

3.4. Kt qu xc nh trinh t nucleotide

Hnh 3.6. So snh trnh t nucleotide ca gen DREB1 CYS vi XBB, AY802779 v FJ965342

Ti liu chia snh mc tng ng trnh t Bng 3.2. So s ti: wWw.SinhHoc.edu.vn

nucleotide ca gen DREB1 ging u tng CYS vi
XBB, AY802779 v FJ965342
XBB AY802779 CYS FJ965342

XBB
AY802779 CYS FJ965342

100

93,3
100

91,1
99,3 100

99,6
99,7 99,6 100

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XBB.seq AY802779.seq FJ965342.seq CYS.seq 0 Nucleotide Substitutions (x100)

0.4

Hnh 3.7. Hnh cy so snh mc tng ng trnh t nucleotide ca gen DREB1 ging u tng

CYS vi XBB, AY802779 v FJ965342

3.5. So snh trinh t amino acid CYS vi XBB, AY802779 v FJ965342

Hinh 3.8. So snh trinh t amino acid ca CYS, AY802779, FJ965342 v XBB

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Bng 3.4. So snh mc tng ng trnh t amino acid trong protein ca gen DREB1 ging u tng

CYS vi AY802779, XBB v FJ965342

XBB AY802779 CYS FJ965342

XBB
AY802779 CYS FJ965342

100

98,3
100

97,0
97,9 100

98,7
99,6 98,3 100

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XBB.pro AY802779.pro FJ965342.pro CYS.pro 1.3 0 Nucleotide Substitutions (x100)

Hnh 3.9. Hnh cy so snh mc tng ng trnh t amino acid ca gen DREB1 ging u tng CYS

vi XBB, AY802779 v FJ965342

Ti liu chia s ti: wWw.SinhHoc.edu.vn KT LUN V NGHI

1. Kt lun
- a tch chit c DNA tng s ca hai ging u tng Cuc vng Yn Sn v Vng Ngn Sn - Bng phng php PCR, chung ti a nhn v pht hin s c mt ca gen DREB1 hai ging u tng Cuc vng Yn Sn v Vng Ngn Sn di 717bp

- a tch dng gen DREB1 ca hai ging u tng Cuc vng Yn Sn v Vng Ngn Sn
- a xc nh trnh t gen DREB1 ca ging u tng Cuc vng Yn Sn

2. ngh
Cn tip tuc nghin cu v gen DREB1 lm c s thit k vector to cy chuyn gen DREB1 1c kh nng chu hn

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