You are on page 1of 33

I HC THAI NGUYN TRNG I HC S PHM

L TH NGC THNG

TUYN CHN, TACH DONG V XAC NH TRNH T GEN cry1C MA HOA PROTEIN TINH TH DIT CN TRNG B CANH VY T VI KHUN Bacillus thuringiensis subsp. aizawai PHN LP T MT S MU T TNH THAI NGUYN

CHUYN NGNH SINH HC THC NGHIM

NGI HNG DN KHOA HC: PGS.TS NG NH BNH

CU TRC CA BAO CAO


1. M U 2. TNG QUAN TI LIU 3. VT LIU V PHNG PHP 4. KT QU V THO LUN 5. KT LUN V KIN NGH

M U
T VN
Bt l vi khun Gram dng, c kh nng sinh cc protein tinh th, gy c mt cch c hiu vi cc cn trng thuc cc b nh b Cnh vy (Lepidoptera), b Hai cnh (Diptera), b Cnh cng (Coleoptera) . Cc protein tinh th ny c m ha bi cc gen cry nm trn cc plasmid. Bt v cc ch phm c ngun gc t vi khun Bt c s dng rng ri nh mt loi thuc tr su sinh hc.

Bacillus thuringiensis subsp. aizawai (Bta) l mt trong nhng 82 di loi ca vi khun Bt c kh nng sinh nhiu loi protein tinh th c c tc dng dit cn trng thuc nhiu b khc nhau. Protein tinh th c Cry1C l mt trong nhiu protein do di loi ny sinh ra c hot tnh chng li cn trng b Cnh vy rt mnh. Vic sng lc v tuyn chn cc di loi Bt c kh nng tiu dit nhiu loi cn trng ch, nghin cu thm v trnh t cc gen cry l hng nghin cu c nhiu nh khoa hc quan tm. T nhng l do trn chng ti chn ti Tuyn chon, tach dong va xac inh trinh t gen cry1C ma hoa protein tinh th dit cn trung b canh vy t vi khun Bacillus thuringiensis subsp. aizawai phn lp t mt s mu t tinh Thai Nguyn.

MC TIU V NI DUNG NGHIN CU


1. Mc tiu nghin cu Tuyn chn c cc chng Bta c hot tnh dit su xanh da lng (Spodoptera exigua) v su t (Plutella xylostella) cao. Xc nh c trnh t gen cry1C t cc chng Bta tuyn chn.
2. Ni dung nghin cu Thu thp cc chng vi khun thuc nhm Bacillus cereus phn lp ti mt s a im ti Thi Nguyn. Phn loi cc chng Bacillus thuringiens bng phng php huyt thanh hc. nh lng nng bo t ca cc chng Bta phn lp c. Sng lc cc chng Bta c hot tnh dit su xanh da lng (Spodoptera exigua) v su t (Plutella xylostella) cao. Pht hin gen cry1C t cc chng Bta c hot tnh dit su cao. Tch dng, c trnh t gen cry1C v so snh vi cc trnh t cng b trn Gene Bank

TNG QUAN TI LIU


1.1. Lch s nghin cu, ng dng vi khun Bacillus thuringiensis 1.2. Nhng c im ca vi khun Bacillus thuringiensis 1.3. c t ca vi khun Bacillus thuringiensis 1.3.1. c t Cry 1.3.2. c t Cyt 1.3.3. c t Vip 1.3.4. C ch tc ng ca protein c t tinh th 1.3.5. Cu trc ca cc nhm c t tinh th 1.4. Gen m ha protein c t tinh th 1.4.1. V tr ca cc gen m ha c t ca vi khun Bacillus thuringiensis 1.4.2. Phn nhm gen m ha c t 1.5. Tng quan v di loi Bacillus thuringiensis subsp. aizawai v gen Cry1C 1.6. Tng quan v cn trng th nghim

Lch s nghin cu vi khun Bacillus thuringiensis

Protein tinh th c t dit cn trng (insecticidal crystal protein)

Cry tree Cy phn loi c t tinh th ca Bt

C ch tc ng ca protein tinh th c

5. Tng quan v di loi Bta v gen cry1C

5.1. Mt s c im ca di loai Bta


Thuc typ huyt thanh H7 theo phn loi ca Bonnefoi v Barjac Sinh tinh th dng lng thp v cu

Hot tnh dit cn trng b cnh vy


Mang mt s gen thuc nhm cry1 nh: cry1C, cry1D, cry1Aa, cry1Ab c s dng ph bin trong nhiu ch phm sinh hc, c chng minh l an ton 5.2. Gen cry1C M ha protein tinh th c khi lng 130kDa Kch thc khong 3kb on bo th m ha c t c kch thc khong 288bp c mt s nghin cu trn th gii v gen cry1C: tch dng, biu hin, chuyn gen. Cc cng b Vit Nam lin quan n gen cry1C cn hn ch

Mt s gen cry1C c cng b trn trang ch ca Crickmore


Name Cry1Ca1 Cry1Ca2 Cry1Ca3 Cry1Ca4 Cry1Ca5 Cry1Ca6[1] Cry1Ca7 Cry1Ca8 Cry1Ca9 Cry1Ca10 Cry1Ca11 Cry1Ca12 Cry1Ca13 Cry1Ca14 Cry1Cb1 Cry1Cb2 Cry1Cb3 Cry1Cb-like Acc No. CAA30396 CAA31951 AAA22343 CAA01886 CAA65457 AAF37224 AAG50438 AAM00264 AAL79362 AAN16462 AAX53094 HM070027 HQ412621 JN651493 M97880 AAG35409 ACD50894 AAX63901 Authors Honee et al Sanchis et al Feitelson Van Mellaert et al Strizhov Yu et al Aixing et al Chen et al Kao et al Lin et al Cai et al Shu et al Li & Luo Li Yuhong Kalman et al Song et al Huang et al Thammasittirong et al Year 1988 1989 1993 1990 1996 2000 2000 2001 2003 2003 2005 2010 2010 2011 1993 2000 2008 2005 Bt LB-R-78 Bt LTS-38 Bt galleriae HD29 Bt c001 Bt 087 Bt TA476-1 insufficient sequence No NCBI link Jan 12 DNA sequence only Source strain Bt entomocidus 60.5 Bt aizawai 7.29 Bt aizawai PS81I Bt entomocidus HD110 Bt aizawai 7.29 Bt AF-2 Bt J8 Bt c002 Bt G10-01A Bt E05-20a Bt C-33 No NCBI link Jan 12 Comment

Tng quan v cn trng th nghim


Su t (Plutella xylostella) Tn khoa hc Plutella xylostella Curtis, cn c tn khc l Plutella maculipennis Curtis, thuc h Yponomeutidae, b Lepidoptera (Cnh vy). Pht trin hu ht cc quc gia trng rau ci trn th gii, ph hoi trn rt nhiu loi rau ci khc nhau nh ci bp, ci b xanh, ci b trng, ci ngt, ci bng. Ngoi ra, su cn gy hi trn mt s loi cy h C nh khoai ty, c chua... Vng i ca su khong 21 40 ngy Su xanh da lng (Spodoptera exigua) Tn khoa hc l Spodoptera exigua Hubner, thuc h Noctuidae (Ngi m), Lepidoptera (b Cnh vy). L i tng gy hi chnh trn u nnh, u xanh, hnh, t v mt s loi hoa mu ngn ngy khc. Vng i ca su khong 30 n 40 ngy

VT LIU V PHNG PHAP


1. Vt liu - 150 chng vi khun thuc nhm Bacillus cereus phn lp t mu t Thi Nguyn, cha qua phn loi - Cc chng vi khun E.coli - Kit huyt thanh min dch chun - Cc cp mi: cp mi c hiu gen cry1C, cp mi M13 - Vect tch dng pGEM-T Easy - Su t (Plutella xylostella) v su xanh da lng (Spodoptera exigua) (cn trng th nghim). - Enzym: enzym gii hn T4-DNA ligase, EcoRI, taq- polymese...ca hng Fermentas - Cc ha cht, thit b my mc phc v cho cc nghin cu trong ti do phng DTVS cung cp. 2. Cc th nghim c tin hnh ti Phng DTVS, Phng TN trng im Cng ngh Gen, Vin Cng ngh Sinh hc

M HNH TH NGHIM Thu thp cc chng Bt cha qua phn loi

Sng lc cc chng Bt sinh tinh th dng trn v thoi nh Phn loi di loi bng type huyt thanh chun Th hot tnh vi cn trng th nghim Phn lp phn on gen cry1C bng PCR

Tch dng gen v xc nh trnh t

So snh vi cc trnh t cng b trn Gene Bank

2. Phng php nghin cu


2.1. Nhom cac phng phap vi sinh, hoa sinh hoc -Nui cy vi khun trn mi trng lng, c -Xc nh nng bo t -Th hot tnh dit cn trng 2.2. Nhom cac phng phap sinh hoc phn t -Phng php tch Plasmid

-Phng php PCR


-Phng php tch dng gen -Phng php xc nh trnh t gen

KT QU V THO LUN
1. Kt qu tuyn chn chng vi khun Bta mang gen cry1C c hot tnh dit su t (Plutella xylostella) v su xanh da lng (Spodoptera exigua) 1.1. Kt qu phn loi hnh dng tinh th ca cc chng Bt - 150 chng vi khun thuc nhm Bacillus cereus c hot ha trn mi trng MPA, trong 3 ngy nhit 280C

Hnh dng khun lc vi khun Bacillus thuringiensis trn mi trng MPA sau 72 gi nui cy 280C

Hnh dng bo t v tinh th ca chng TN1.12 v TN 36.3 phng i 100X di knh hin vi quang hc (vt knh du)

- S chng Bt sinh tinh th: 54/150 chng


Kt qu phn loi hnh dng tinh th cc chng Bt phn lp STT Hnh dng tinh th S lng chng T l (%)

1
2 3

Lng thp
Cu Lp phng

21
4 1

39
7 2

4
5 6

Lng thp, cu
Lp phng, cu Khng xc nh

26
1 1

48
2 2

- S phong ph v dng tinh th ca cc chng Bt - 26 chng sinh 2 loi tinh th lng thp v cu c s dng tin hnh phn loi di loi

1.2. Kt qu phn loi di loai bng phng phap huyt thanh hoc -Di loi Bta thuc typ huyt thanh H7 -S dng b X huyt thanh phn loi

Hnh nh ngng kt ca chng 4J4 (A) v TN 6.12 (B) vi typ huyt thanh H7 di knh hin vi quang hc 1: trc khi nh huyt thanh 2: sau khi nh huyt thanh

Kt qu phn loi di loi ca cc chng Bt sinh tinh th


STT 1 Di loi Bacillus thuringiensis toguchini Typ huyt thanh 31 1 S chng ngng kt T l (%) 3.84 38.46 7.69 3.85 19.23 3.85 3.85 7.69 7.69 3.85 11 Tng 9 26 100

2
3 4 5 6 7 8 9 10

aizawai
tochigiensis asturiensis thuringensis sotto tolworthi canadensis kim Khng ngng kt

7
19 53 1 4 9 5 52

10
2 1 5 1 1 2 2 1

1.3. Kt qu th hot tnh dit su ca cac chng Bta nghin cu Nng bo t ca cc chng Bta nghin cu: 2,5 5.109 bo t/1ml 1.3.1. Kt qu th hot tnh ca cc chng nghin cu vi su xanh da lng (Spodoptera exigua)

Hnh nh th hot tnh dit su xanh da lng (Spodoptera exigua) ca cc chng Bta nghin cu

Kt qu th hot tnh su xanh da lng (Spodoptera exigua)

sau 3 ngy th nghim


T l su cht (%) STT 1 2 3 4 5 6 7 Tn chng C m C dng (4J4) TN 1.12 TN 3.4 TN 4.4 TN 5.3 TN 6.12 Nng 107 bo t/ml 0 Nng 109 bo t/ml 0

33,33
22.22

77,78
55.56

11.11
11.11 11.11 22.22 22.22 22.22

22.22
33.33 44.44 55.56 66.67 55.56

8
9

TN 28.6
TN 36.3

1.3.2. Kt qu th hot tnh ca cc chng nghin cu vi su t (Plutella xylostella)

Hnh nh th hot tnh dit su t ca cc chng Bta nghin cu

Kt qu th hot tnh dit su t (Plutella xylostella) ca cc chng Bta sau 3 ngy th nghim
- 7 chng Bta nghin cu u c hot tnh vi c 2 loi

cn trng th nghim
STT Tn chng

T l su cht (%)
5 7

Nng 10 Nng 10 - Hot tnh dit su ca cc chng t l thun vi nng bo t/ml 0 bo t/ml 0

bo 1 c s dng v thi gian th hot tnh. t C m


2 3 4 5 6 7 8 9 C dng (4J4) TN 1.12 TN 3.4 TN 4.4 TN 5.3 TN 6.12 TN 28.6 TN 36.3 76,67

- Cc chng TN 1.12, TN 6.12, TN28.6 v TN 36.3 c hot


93,33 80,00 83.33 76.67 80,00 80,00 86.67 83.33 53.33 63.33 50,00 53.33 56.67 60,00 53.33

tnh dit cao i vi c 2 loi cn trng th nghim

1.4. Kt qu phat hin gen cry1C bng phng phap PCR vi cp mi c hiu -S dng cp mi c hiu TYIC v TYIUNI -on gen theo tnh ton l thuyt c kch thc:288bp
11 22 3 44 5 66 7 7 8 8 M 3 5 M

300 bp

Hnh nh in di sn phm PCR ca cc chng Bta nghin cu Ging 1, 2, 3, 4, 5, 6, 7, 8: TN 1.12, TN 3.4, TN 4.4, TN 5.3, TN 6.12, TN 28.6, TN 36.3, C dng M: DNA Marker

2. Kt qu tch dng v xc nh trnh t gen cry1C

2.1. Kt qu tach dong gen cry1C


2.1.1. Gn sn phm PCR vo vector tch dng pGEm T Easy 2.2.2. Bin np vector ti t hp vo vi khun E. Coli DH5

Khun lc xanh v trng trn a thch LBA sau 14 gi nui cy

600 bp 300 bp

Hnh 3.8. Kt qu in di sn phm PCR vi mi M13 1,2,4: PCR cc dng khun trng ca cc pasmid ti t hp pGEM-T Easy cry1C TN28.6 - p3, pGEM-T Easy cry1C TN 36.3 - p1 v pGEM-T Easy cry1C TN 6.12 p7 3: PCR dng khun xanh (C) 4: Thang DNA chun

1 1

22

33

4 4

M5

3000 bp

300 bp

Hnh 3.9. in di sn phm ct DNA plasmid tch chit t cc dng khun lc trng trn agarose 1% Ging 1,2,4. Sn phm ct DNA plasmid dng khun lc trng TN28.6 p3, TN 36.3 - p1 v TN 6.12 p7 Ging 3. DNA plasmid dng khun lc xanh (C) Ging 5.Thanh DNA chun

300 bp

Hnh 3.10.Kt qu sn phm PCR gen cry1C t cc DNA plasmid hp pGEM-T Easy cry1C TN28.6 - p3, pGEM-T Easy cry1C TN 36.3 - p1 v pGEM-T Easy cry1C TN 6.12 p7

2.2. Kt qu xc nh trnh t gen cry1C Kt qu so snh vi trnh t c m s AF362020.1 trn Gene Bank

Gen cry1C ca 3 chng tch dng: TN6.12, TN28.6, TN36.3 u thuc phn nhm gen cry1Ca

KT LUN V NGH
KT LUN 1. T 150 chng vi khun thuc nhm Bacillus cereus cha qua phn loi, xc nh c 54 chng Bt sinh tinh th, trong 26/54 chng sinh 2 loi tinh th dng lng thp v cu. 2. phn loi c 26 chng Bt sinh tinh th lng thp v cu bng 56 typ huyt thanh chun. 26 chng Bt thuc v 9 di loi khc nhau, trong ph bin l cc di loi aizawai, thuringiensis, trong c 10 chng thuc di loi aizawai, chim t l 38,46%. 3. sng lc c 7 chng Bta c hot tnh dit su t (Plutella xylostella) v su xanh da lng (Spodoptera exigua), trong 4 chng TN1.12, TN6.12, TN28.6 v TN 36.3 c hot tnh dit su cao nht. 4. khuch i c gen cry1C t 7 chng Bta c hot tnh dit su bng cp mi c hiu. Sn phm PCR thu c c kch thc 288 bp. 5. tch dng v xc nh trnh t gen cry1C ca 3 chng Bta v so snh vi trnh t c m s AF362020.1 c cng b trn Gene Bank. Trnh t c so snh c tng ng 100% vi on gen cry1C ca 2 chng TN6.12 v TN28.6 v 99, 65% i vi on gen cry1C ca chng TN36.3. Cc gen tch dng c thuc phn nhm gen cry1Ca.

NGH 1. xc nh c trnh t on gen cry1C ca cc chng TN 36.3, TN 6.12 v TN 28.6 c hot tnh dit su cao. Trn c s c th thit k cp mi khuch i ton b gene cry1C tin hnh biu hin protein Cry1C trn vi khun E.coli. 2. Thit k vector biu hin gen cry1C trong vi khun thuc nhm Bacillus khng sinh tinh th.

You might also like