Professional Documents
Culture Documents
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
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.
C ch tc ng ca protein tinh th c
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
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)
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
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
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
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
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
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.