TRNG I HC PHNG NG KHOA CNG NGH SINH HC V MI TRNG

TRNG I HC PHNG NGKHOA CNG NGH SINH HC V MI TRNG

N TT NGHIP

TI: : ng dng ca ch th phn t xc nh dng b m phc v cho chn to ging khoai ty khng bnh mc sng

GVHD: TS. L Hng Lnh SVTH : V Th Phng Tho MSSV: 510301028

NI DUNG CHNHMc ch v ni dung nghin cuKt lun v nghChng 3: Kt qu v tho lunChng 1: Tng quan ti liuChng 2: Vt liu v phng php nghin cu

Mc ch v ni dung nghin cu

Mc chXc nh c b m mang gen khng bnh mc sng ca khoai ty bng phng php s dng cc ch th phn t.Ni dung Phn tch ch th phn t a hnh gia cc ging/dng khoai ty dng lm b m phc v phn tch kiu gen trong qun th.

CHNG 1: TNG QUAN TI LIU1.1. Gii thiu chung v cy khoai tyCy khoai ty tn khoa hc Solanum tuberosumL Gii : Plantae. B : Solanales. H : Solanaceae. Chi : Solanum. Loi : S.tuberosum

Cy khoai ty l loi cy nng ngip, trng ly c cha tinh bt rng ri nht th gii.

Tnh hnh sn xut cy khoai ty Vit NamCy khoai c ngi Php mang n Vit Nam t hn 100 nm trc yHin nay, 70% din tch khoai ty tp trung cc tnh ng bng Sng Hng v khoai ty tr thnh cy trng v ng quan trng ti y. Vit Nam, cy khoai ty c trng trn din tch 40.000 ha v sn lng t 440.000 tn/nm. Sn lng ny mi ch p ng c 20 n 25% nhu cu tiu dng trong nc Tim nng ca cy khoai ty nc ta cn rt ln.

1.2. Bnh mc sng cy khoai tyTc ly lan v pht trin rt nhanh

Tc hi Bnh mc sng gy thit hi nng sut ln n 70%

Bnh mc sng khoai ty gy bi loi nm c tn khoa hc Phytopthora infestans, thuc h Pythiaceae

Sau 2-3 tun cy nhim bnh c th cht hon ton

L mt trong nhng bnh nghim trng nht trong lch s nhn loi i vi cy khoai ty

Triu chng ca bnh mc sng vi cy khoai ty

Triu chng trn l khoai tyNhng vt thng ban u rt nh c kch thc t 2-10 mm v hnh thi c th khc nhau, phn b tn thng do bnh ngy cng ln ln v to thnh cc vng trn ng tm ca vt bnh trn l.Triu chng trn cnh, thn Thn cy b tht li L phn pha trn thn b hoVt bnh c mu sc t sng n ti to nn cc vng trn trn thnKhi b nng thn cy d dng b gy. Triu chng trn c C b bnh c vt bnh hnh a mu sc nu en to nn hoa vn m trn cMu sc c b bin mu. C b nhim bnh tr nn cng, kh v chc li.

ng dng ch th phn t trong chn to ging khoai ty khng bnh mc sng.Cc ch th phn t nh AFLP, RAPD, SSR c s dng rng ri trong cc nghin cu chn ging v di truyn ca khoai tyS dng ch th SSR cho php ti hin kt qu tt hn v mc a hnh cao hn so vi ch th RAPDHin nay, mc biu hin a hnh trong phn tch AFLP cao hn so vi phn tch SSR. Tuy nhin, SSR l ch th ng tri, vic thc hin phn tch n gin hn, khng cn cc thit b phc tp nh phn tch AFLP. Nh vy s dng ch th SSR l u vit nht

CHNG 2: VT LIU V PHNG PHP NGHIN CU

Vt liu nghin cu Vt liu nghin cu gm 10 ging nhp ni (Ging s 47; Ging s 106; Ging s 113; Ging s 115; Ging s 116; Ging s 119; Ging s 124; Ging s 129; Ging s 140; Ging SOLARA) v 2 ging KT3, PO3 ang c trng i tr a phng.8 ch th phn t SSR c s dng xc nh ch th a hnh gia vt liu b m trong nghin cu

Cc cp mi SSR dng trong nghin cu khoai ty

S TTTn miTrnh tKch thc on gen nhn bn, bpNhit gn mi thc t, oC1STI 0006F: ctttagtccttggcagagcttR: cgggctgattcttcttcatc200-250562LP6F:tgcctactgcccaaaacat R:actggctgggaagcatacac128-166573Pota6F: tattcccccttcctactcaaR: tcttccacattcctaacctg202554Pota 16F: ttctgatttcatgcatgtttccR: atgtgtggtctacaaaaagggg236605Pota 48F: ttgcgtgaagcagccgtaaaR: gcccagtaagtaaaacattg110546Pota 40F: ttcaaagaaatcacaaaatcR: tcaagaccccaaaactaact145517Pota 43F: aacattacaacacattagcaR: aacttatctgaaactctcgt204528Pota 45F: attctccgaaagtatgtctR: tgggatgagttggtgtgtc450522.2. Phng php nghin cu

1. Tch DNA tng s bng phng php CTAB2.Xc nh nng v tinh sch ca DNA tng s bng phng php quang ph k3. Kim tra nguyn vn ca DNA tng s bng phng php in di trn gel agarose4.Tin hnh phn ng PCR5. Phn tch kt qu PCR bng k thut in di trn gel polyacrylamide

CHNG 3: KT QU V THO LUN

1.Kt qu kim tra DNA tng s tch chit theo phng php CTAB trn gel agarose 0,8%

Cc mu DNA khng b t gy, vic loi b ARN bng RNase tin hnh kh tt th hin cc bng in di gn, r. Nhng mu DNA ny iu kin s dng cho th nghim sinh hc phn t tip theo.

2. Nng v ch s tinh sch ca DNA tng s Mu Nng (ng/l)Ch s tinh sch (A)260/(A)280 47560,01,80 106908,71,90 1131096,11,92 1151269,61,92 116849,51,91 119731,71,88 124832,11,90 1291114,81,92 140782,01,80 SO699,11,85 KT3977,11,90 PO31112,11,85 Kt qu tch chit DNA cho thy tt c cc mu DNA u c ch s tinh sch nm trong khong 1,8 2,0 nh vy 100% s mu DNA tinh sch,

3. Kt qu thu thp pht trin ch th v tm nhit gn mi c hiu ca phn ng PCRKt qu chng ti xc nh v chn lc c 8 ch th phn t SSR s dng trong nghin cu xc nh ch th a hnh gia dng b m. xc nh c nhit gn mi c hiu cho 8 cp mi nghin cu

4. Kt qu kho st a hnh gia cc ging cho v nhn QTL/gen khng bnh mc snga hnh gia hai ging/dng khoai ty c th c pht hin bng chiu di khc nhau ca cc on lp li c khuych i bi phn ng PCR khi s dng cng mt cp mi SSR. Trong nghin cu di truyn tnh khng bnh mc sng khoai ty chng ti s dng 8 ch th phn t trn cc nhim sc th khc nhau xc nh ch th phn t a hnh gia 12 dng/ging b m khc nhau, phc v cng tc lai to chn lc ging khngCH TH PHN T STI0006

Kt qu a hnh r rt gia cc gingSn phm gen nhn bn c kch thc nm trong khong 200-250bp. S lng allen xut hin cc ging nghin cu nm trong khong t 1 n 4. S Allen xut hin nhiu nht l cc Ging s 47, Ging s 115, Ging s 119, Ging s 124 v Ging KT3 vi 4 allen. S Allen xut hin t nht l Ging PO3 vi 1 allen. Cc ging cn li c t 1 n 3 allen

Chia 12 ging nghin cu thnh 5 nhm phn bit: Nhm 1 gm 5 ging khng a hnh vi nhau l: Ging s 47; Ging s 115; Ging s 119; Ging s 124; v Ging KT3. Nhm 2 gm 2 ging khng a hnh vi nhau l: Ging s 116 v Ging s 129. Nhm 3 gm 2 ging khng a hnh vi nhau l: Ging s 140 v Ging SO. Nhm 4 gm 2 ging khng a hnh vi nhau l: Ging s 106 v Ging s 113. Nhm 5 ch c Ging PO3. Nh vy, Ging PO3 a hnh vi tt c cc ging cn li

CH TH PHN T LP6Gen nhn bn c kch thc khong 128-166 bp, s bng gen phn tch nhiu (t 3 n 10 bng gen). S allen nhiu nht l Ging s 140 vi 10 allen Ch c 2 Ging s 116 v Ging s 129 khng a hnh vi nhau. Tt c ging cn li u a hnh vi nhau v a hnh vi Ging s 116 v Ging s 129.

CH TH PHN T POTA 16Gen c nhn bn c kch thc 236 bp v s allen ca tt c cc ging u bng 2

Kt qu phn thnh 3 nhm ring bit Nhm 1 gm 5 ging khng a hnh vi nhau l: Ging s 106, Ging s 119, Ging s 124, Ging s 129, v Ging s 140. Nhm 2 gm 6 ging khng a hnh vi nhau l: Ging s 47, Ging s 113, Ging s 115, Ging s 116, Ging SO, v Ging KT3. Nhm 3 ch c 1 ging PO3. Nh vy ging PO3 a hnh vi tt c ging cn li.

CH TH PHN T POTA 6 Sn phm PCR c kch thc 202 bp. S Allen cc ging thay i trong khong t 2 n 8. Trong , Ging s 47 v Ging s 106 c s allen nhiu nht vi 8 allen. Ging c s allen t nht l Ging s 113, Ging s 119, Ging s 124, Ging SO v Ging PO3 vi 2 allen

12 ging nghin cu c th phn ra thnh 5 nhm khc nhau v c tnh di truyn. Nhm 1 gm Ging s 47 v Ging s 106 khng a hnh vi nhau. Nhm 2 gm 5 ging khng a hnh vi nhau l Ging s 113, Ging s 119, Ging s124, Ging SO, v Ging PO3. Nhm 3 gm Ging s 115 v Ging s 116 khng a hnh vi nhau. Nhm 4 gm Ging s 140 v Ging KT3 khng a hnh vi nhau. Nhm 5 ch c mt Ging s 129. Nh vy, Ging s 129 a hnh vi tt c ging cn liCH TH PHN T POTA 48Kt qu gen nhn bn vi ch th POTA48 c kch thc 110 bp v s allen thay i t 1 n 2. Ging s 116 v Ging SO c s allen nhiu nht vi 2 allen

Kt qu phn thnh ba nhm ring bit. Nhm 1 gm 9 ging khng a hnh vi nhau l Ging s 47, Ging s 113, Ging s 115, Ging s 119, Ging s 124, Ging s 129, Ging s 140, Ging KT3, v ging PO3. Nhm 2 gm 2 ging l Ging s 116 v Ging SO. Nhm 3 ch c Ging s 106. Nh vy, Ging s 106 cho kt qu a hnh vi tt c ging cn liS alen a hnh v gi tr PIC ca cc ch th phn t SSR phn tch vi 12 ging khoai tySTTCh th SSRS allen a hnhTng s allen a hnhCh s a hnh PIC1STI00064370.742POTA68460.823POTA163240.644LP614730.915POTA483140.36Tng32194Trung bnh6,438,80,694Gi tr ch s a hnh PIC bng 0 ti v tr locus ch c 1 allen n hnh. Cc ch th SSR c gi tr PIC ln hn hoc bng 0.5 s cho s phn bit cao v t l a hnh ca ch th . Nh vy, qua bng trn ta nhn thy c 4/5 ch th c gi tr PIC > 0.5. iu ny khng nh rng, 80% s ch th s dng trong nghin cu c s khc bit ln v t l a hnh.

KT LUNTch chit DNA khoai ty bng phng php CTAB cho hiu qu cao, tch chit c 12 mu DNA khoai ty tinh sch, cht lng tt.S dng 8 ch th phn t nhng ch c 5 ch th phn t s dng nhn cc on DNA trong 12 ging khoai ty u cho a hnh cao v c th phn bit s khc bit gia cc gingChn c 2 ging khoai ty l ging s KT3 v 116 dng lm b m phc v cho chn to ging khoai ty khng bnh mc sng.

NGHTrong th nghim ny ti nghin cu c 5 ch th phn t xc nh dng b m phc v chn to ging khoai ty khng bnh mc sng, trong thi gian ti ti s tin hnh nghin cu thm nhiu ch th phn t hn na xc nh dng b m phc v chn to ging khoai ty khng bnh mc sng.Cm n thy c v cc bn lng nghe