TAP CHi ICHOA HQC TRCfltNG BAI HQC H 6 N G BtTC - S6 40.2018
iTNG DUNG CHI THI PHAN Tif ADN TRONG CHQN TAG GIONG CA CHUA CHIN CHAM CHtTA GEN RIN
Nguyen Thi Vin', Nghigm Thj Hmmg^
TOM TAT
Nghiin cuu ndy sir dung chl thi phdn ttr ADN di chon loc cdc cd the tru tti trong qudn the phdn ly F2 cd chua gen rin (ripening inhibitor) kiem sodi ddc tinh chin chdm, co ndng sudt cao vd chdt luang tdt a cd chua. Thi nghiem thtrc hiin tren qudn the phdn ly cd ngudn goc tir gidng cd chua lai cd md sd 159 nhap noi lir My bai Trung tdm Bdo ton vd Phdt trien ngudn gen cdy trdng.
Tir khoa: Gen chin chdm, cd chua chin chdm, gen rin.
l . D A T V A N D E
Cdy ca chua (Licopersicum esculentum Mill) Id loai rau an qua cd gid trj dinh dudng cao, duge trdng vdi dien tich Idn nhdt trong cac cdy rau. Sdn xudt ca chua d midn Bdc nude ta chu yeu d vu Ddng Xudn, thdi didm cd nhidu ydu id mdi trudng thu$n lgi cho ca chua sinh trudng, phdt trien vd it bi sdu benh phd hgi nen ndng suit vd chit lugng khd cao. Tuy nhidn do thu hoach tdp tmng nen gid tuong ddi thip, anh hudng ddn thu nhap cua ngudi sdn xuit. Trong khi do, tii thdng 6 - 9 khdng cd du cd chua cung c i p cho thi tmdng. Vi thd, da cd nhieu bi^n phdp kf thuat nhdm rai vy ca chua nhung cac gidng mdi chgn tao ciia ta hien nay chua cd gidng nao cd ddc tinh chin cham ciia qud.
Hien nay, tren the gidi da cd nhidu nghien ciiu chgn tao thdnh cong cde gidng cd chua chin chdm cd ddc didm qua dat dil kich thudc va tich luy dii chit khd nhung ehul rdt chdm do mdt hoac han chd khd ndng tdng hgp ethylene - la mOt hormone tin hi$u hogt hda mgt sd enzyme lien quan ddn qua ttinh chin cua qud. Cdc gidng cd chua nay cd chiia cac gen dOt bidn ty nhien lidn quan ddn dac tinh chin chdm. Trong dd, rin Id mgt dot bidn lan ttai thiy d cdy ca chua ttdng tu nhidn da duge sii dung ttong nhidu chuong trinh chpn tao gidng cd chua chin chdm. Chi thi phdn tii ADN lien kdt chdt vdi gen nay ciing da duge phat tridn vd sil dyng (Xiaoli, 2010). Tuy nhien, hien nay d Vi$t Nam vdn chua tao duge gidng ca chua nao cd dac tinh chin cham. Vi vay ling dyng chi thi phan hi nhdm phdt hi?n gen chm cham - rin nhdm tao ngudn vdt lieu cho cdng tac chpn tgo gidng ca chua chin cham la vi$e lam cin thidt.
2. NOl DUNG
2.1. Vdt lifu v i phirong phdp nghien ciiu 2.1.1. Vdlliiu, thai gian vd dia diim nghiin cuu
" Giang vien thoa Nong - Lam - Ngu nghidp, Tmdng Dgi hoc Hdng Due
T ^ CHI KHOA HQC T R t / ^ N G B.»I HQC H 6 N G DJSC - S 6 40.2018
Vat lieu: Quin thi phan ly cua gi6ng ca chua chin cham Fl duac nhap ngi tir My boi Trung tam B^o t6n va Phdt triln ngu6n gen cay trong, c6 ma s6 la 159; GiSng doi chung HT152. Su dung cap m6i PCR dimg d6 phat hien cac ca the mang gen chin chSm rm (Xiaoli, 2010):
Fw: 5 ' -TTAAGTTGCGAAGAACTTGGTGTTACCTT-3' Rv: 5' -GCCAAAACACTTCAATTTCCTTTAAAAGTT-3'.
Dia diem nghien ciiu: thuc hien tai phong thi nghidm bp mon Sinh hpc phan hi va Cong nghe sinh hoc ung dung - khoa Cong nghp sinh hpc - Hpc Vien Nong Nghiep Viet Nam, Trau Quy - Gia Lam - Ha Npi.
Thoi gian th\rc hien: tir ngay 08/2015 - 08/2016.
2.1.2. Ngi dung nghiin cuu
Danh gia va chpn loc cac ca the mi tu co nang suat cao, chat lupng t6t, co dac tinh chin cham trong quSn the phan ly cua giong ca chua lai chin cham 159.
Su dyng chi thi phan tu ADN dua tren ky thuat PCR phat hien cac ca th6 mang gen chin cham rin trong quan the phan ly giong ca chua lai chin cham 159.
2.1.3. Phuangphdp nghien ciru
2.1.3.1. Ddnh gid dgc tinh chinchgm cua qud Theo doi moi cay 5 qua ve cac chi tieu sau:
Thai gian tu khi qua dat kich thuoc toi da den khi chuyin mau;
Thai gian tOr khi qua chuySn mau den khi chin hoan toan;
Thcri gian tiJr khi qua chuyin mau den khi hong (thai gian bao quan).
2.1.3.2. Su dyng chl thi phdn tir ADN (PCR) phdt hiin gen chin chgm rin Hda chdt vd dem chiit
DEB (DNA Exh-action Buffer): 2% CTAB (w/v), 1,5 M NaCl, 1 OOmM Tris- HCI, 20 mM EDTA, 2% PVP (w/v), 2% p- Mercaptoethanol. Hoa tan CTAB va cac hoa chdt khac ti-ong nuoc cSt 2 15n bang each u trong waterbath 6 65''C din khi CTAB tan hoan toan.
Them 2% p- Mercaptoethanol ngay truac khi sir dung.
TE buffer: lOmM Tris-HCl, ImM EDTA, pH 8.0.
Hoa chdt khac: Phenol : Chloroform : isoamyl alcohol (25:24:1), Chloroform:
isoamyl alcohol (24:1), 100% ethanol, 76% ethanol.
g«y trinh tach chiet ADN tir Id cd chua
Quy trinh tdch chiet ADN tu la ca chua duoc tien h&nh theo phuang phap CTAB (Doyle va Doyle, 1990).
Ky thugt PCR dung di phdt hi$n gen chin chdm
Trinh tu moi diing phat hi?n gen chin ch|im rin (Xiaoli, 2010) Fw: 5' -TTAAGTTGCGAAGAACTTGGTGTTACCTT-3' Rv:5' -GCCAAAACACTTCAATTTCCTTTAAAAGTT- 3 '
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TAP CHi KHOA HQC TRgpNG BAI HQC H 6 N G BtTC - S6 40.2018
Thanh phin phdn img PCR: Theo hudng ddn cua cdng ty KAPA BIOSYSTEMS cho tdng thd tich 25yl gdm: PCR buffer (5X) Syl, MgCb (25mM) 3nl, dNTPs (lOmM) 0.2^1, Forward primer (lOjiM) 1.25yl, Reverse primer (lOuM) 1.25yl, KAPATaq HotStart DNA Polymerase (5U/yl) O.lyl, DNA khudn lyl.
Chu ky nhiet cho phan ling PCR phdt Men gen rin: 94°C ttong 4 phdt; 35 chu ky:
94°C ttong 30 gidy, 53°C trong 1 phiit, 72°C ttong 1 phiit vd kdt thiic Id 72°C trong 10 phut.
Sdn phdm PCR duge phdn tach ttong bd didn di su dung gel agarose 1% ttong d$m TAE (IX), nhudm bdng ethidium bromide vd quan sdt va chyp anh duoi den UV.
2.1.4. Xirlysd lieu
Sd li^u duge xii ly bdng phan mdm Excel 2007.
2.3. K^t qua vd thdo luan
2.3.1. Ddnh gid thdi gian bdo qudn ciia qud cd chua chin chdm Kdt qud ddnh gid thdi gian bdo qudn qud cd chua the hien d bdng 1.
Bang 1. Danh gid dac tfnh chin cham vdi qud ciia cdc cd t h i nghien ciiu
Cdthd
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Thdi gian tir khi qud Dgt kieh thudc tdi da
den khi chuyen man (ngdy) 14,6±1,3 12,8±0,8 12,6±0,9 13,2±1,1 13,4±0,9 12,4±0,9 12,6±0,5 12,8±1,8 11,2±0,8 13,4±1,5 12,2±0,8 12,4±0,9 12,0±I,0 12,0±0,7
Chuyen mdu ddn khi chin hodn toan (ngay)
12,2±1,3 12,4±1,9 12,4±0,5 10,0±0,7 15,2±0,8 8,6±1,9 8,6±1,3 13,2±2,6
9,8±0,8 15,2±1,3
8,0±0,7 13,2±2,8 11,8±1,6 12,2±1.8
Chuyen mdu den khi bi hdng (ngdy)
46,0 35,0 31,0 32,0 36,0 30,0 29,0 54,0 28,0 40,0 31,0 28,0 31,0 33,0
TAP c m KHOA HQC TRircUNG BAI HQC HONG BljC - S 6 40.2018
16 17 18 19 20 HT152
11,8±0,8 12,8±0,8 12,8d=l,l 12,8±0,8 13,4±0,5 11,8±0,5
6,6±0,5 12,0±1,0 12,4±2,7 8,6±0,5 12,0±1,4
8,8±0,6
25,0 33,0 31,0 28,0 27,0 27,0 2.3.1.1. Th&i gian tie luc qud dgt kich thu&c toi da din khi chuyen mdu
Thai gian tir luc qua dat kich thuac t6i da den khi chuyen man la thoi ky qua khong tang tnrang ve kich thuac ma chu ylu la qua trinh tich liiy chat dinh duong vao qua. Thu hoach qua khi con xanh giup viec van chuyen va bao quan dupe lau hon nhung chat lupng qua khong dam bao, trong khi thu hoach qua khi chin hoan toan dam bao duac chat lupng qua nhung qua nhanh thoi hong do de dap nat trong khi van chuyen. Thtri diem qua chuyen mau la thai dilm thu hai vua dam bao chat lupng qua khi chin, ddng thoi qua vln du dp cimg dam bao khong dap nat trong van chuyen. Vi the theo doi then diem qua chuyen mau giup xac dinh thai diem thu hai qua hpp ly. Ket qua theo doi b bang 1 cho thay, nhung cay CO thoi gian qua dat kich thu6c toi da den khi chuyin mau dai nhat 13,4 ngay d cay so 10 va 14,6 ngay acSy 1.
2.3.1.2. Thai gian tir khi qud chuyin mdu din khi chin hodn todn
Thdi gian tir khi qua chuyen mau den chin hoan toan cho biet thai gian chin cua qua nhanh hay chSm, dac trung cho tinh chin cham cua qua. S6 lieu tir bang 1, giiip chiing ta nhan thdy nhom qua co thai gian chin ngSn (8,6 - 10,8 ngay) b nhiJng ciy 6, 7, 9, 16, 11, 19;
nhom qua co thai gian chm trung binh (10,8 - 13,0 ngay) gom nhiing qua a cay 1, 3, 2, 13, 14, 15, 17, 18, 20; nhom qua c6 thai gian chin c h ^ hon (hi 13,0 - 15,2 ngay) gom nhiing qua or cay 5, 8, 10,12.
3.1.3. Thai gian tic khi qud chuyen mdu den khi bi hong
Thdi gian ton trO qua dac trung cho kha nang bao quan cua ca chua sau khi thu hai.
Qua cang bao quan dupe ISu thi cang co y nghia ve kinh te. Qua bang 1, qua thu dupe tir cac ca thi trong quin thi phan ly co thai gian tir khi qua chuyen mau den khi hong (thdi gian tdn hii) biln dpng tu 25.0 ngay (cay 16) din 54,0 ngay (cay 8), mau qua d cay doi chiing CO thdi gian bao quan 27,0 ngay. Khi phan ra 3 nhom khac nhau dua vao thdi gian ton trQ (thdi gian ton trtt ngfin, trung binh va dai) thi nhom cay co qua tdn tru dupe trong thdi gian trung binh (34,7 - 44,3 ngay) gom cac cay s6 2, 5, 10; nhdm cay cd qua tdn trii dupe trong thdi gian dai nhat (44,3 - 54 ngay) gdm cay 1 (46 ngay); cay 8 (54 ngay); nhdm cdn lai gdm nhung cay co qua ton trii duac trong thdi gian ngdn han (25,0 - 34,7 ngay).
Ddi vdi nhiing cay c6 thdi gian qua ton trii trung binh va dai, cay 1, cay 5 va cay 10 cd qua khi chin mau vang; cdn qua cua cay 2 va cay 8 khi chin hoan toan co mau do.
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T ^ P CHi KHOA HQC TRlTONG BAI HQC HONG PU'C - S 6 40.2018
Khi tiln hanh so sanh thdi gian bao quan trong phong doi vdi 2 mau qua: mhi qua thu dupe tir ca thi s6 8 (qua cd thdi gian bao quan lau nhat) va mau qua thu duac tii gidng ddi chiing HT152 (xem hinh 1), chung toi nh^n thSy qu^ thu dupe d cay so 8 c6 thdi gian bao quan dai han rdt nhieu so vai ddi chung. Ciing trong mpt dieu kien va thdi gian ton trir, mau qua HT152 da thdi riia, trong Ichi mau qu^ d cay so 8 chi co mpt s6 dau hieu bdt ddu bi hdng.
Hinh 1. So sanh thiri gian bSo qudn trong phdng & thin diem 54 ngfky theo doi gifra 1 mlu qua or ci t h i sd 8 va mlu qua d* giong HT152 trong cung di§u kien bao quan 2.3.2. Kit qud PCR sir dyng cgp mdi phdt hiin gen chin chgm
•i*wi»irj>C»*»f*f<n •«r:i»otiJtttiUitl}
lOOObp 7S0bp-
Hlnh 2. Ket qua flif n di su dung cap mhi phat hi^n cii t h i mang gen chin chdm rin Cdc gieng 1-14: cdc cdy duge chpn lpc tii qudn thd phan Iy cila gidng 159, 15 la gidng ddi chirng HT152 khdng chiia gen.
TAP C M KHOA HQC TRlTOWG P A l HQC HONG BlTC - SO 40.2018
Kit qua PCR su dung c^p mdi phat hien gen chin cham rin cho thay cac ca the sd 1, 2,3,4,5, 8, 10, 11, 12, 13, 14, 18, 19 co chua gen vdi kich thudc san phSm la 850 bp. Kit hpp vdi nhiing nhan xet d phdn 2.2.1.3 ddi vdi bang so lieu 1, nhiing ca the nay khi theo ddi thdi gian bao quan qua trong phdng dlu cd thdi gian bao quan dupe tren 31 ngay, lau han so vdi nhiing qua khong chiia gen (tur 25,0 den 30,0 ngay) (bang 1).
4. KET LUAN
Da phat hien dupe cac ca thi sd 1, 2, 3, 4, 5, 8, 10, 11, 12, 13, 14, 18, 19, 20 cd chiia gen rin.
Trong sd cac ca the mang gen chin ch§m rin, ca the so 8 cd thdi gian bao quan dai nhat (han 27 ngay so vdi ddi chung), qua cd mau do, 2 ngan, khdi lupng trung binh 52,2g, cd kha nang ung dung trong san xuat ca chua tai Viet Nam.
De xuat mdt so ca the uu tu cd tiem nSng cho nang suat cao, chat luong tdt, chua gen chin cham rin va thdi gian bao quan dai (hon 34 ngay) gdm ca thi sd 1,2,5,8,10.
TAI LIEU THAM KHAO
[1] B6 Ndng nghiep va Phat trien nong thdn (2006), Gidng cdy cd chua, Quy pham khao nghiem gia tri canh tac va gia tri su dung, 10TCN219: 2006 .
[2] Bp Ndng nghiep va Phat triln nong thdn (2002), Quy phgm khdo nghiem tinh khde biit, tinh dong nhdt vd tinh on dinh cua giong cd chua, 10TCN557-2002.
[3] Lin, Z., Zhong, S., Grierson, D (2009), Recent advances in ethylene research.
Joumal of Experimental Botany, 60(12), 3311-3336
[4] Liu, Y., Roof, S., Ye, Z., Barry, C, van Tuinen, A., Vrebalov, J., Bowler, C, Giovannoni, J (2004), Manipulation of light signal transduction as a means of modifying fruit nutritional quality in tomato. Proc Natl Acad Sei USA, 101(26), 9897-9902.
[5] Mannmg, Tor, M., Poole, M., Hong, Y., Thompson, A. J., King, G. J., Giovannoni, J. J., Seymour, G. B. (2006), A naturally occurring epigenetic mutation in a gen encoding an SBP-box transcription factor inhibits tomato fruit ripening, Nat Genet, 38(8), 948-952.
[6] Martel, C, Vrebalov, J., Tafelmeyer, P., Giovannoni, J. J (2011), The Tomato MADS-Box Transcription Factor RIPENING INHIBITOR Interacts with Promoters Involved in Numerous Ripening Processes in a COLORLESS NONRIPENING-Dependent Manner. Plant Physiology, 757(3), 1568-1579.
[7] Xiaoli (2010), The development of longer shelf-life gen marker and assisted selection of tomato inbredlines. Huazhong Agricultural University, 46.
[8] http: llwww.esalq.usp.br/tomato
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TAP CHl KHOA HQC TRlfOING BAI HQC HONG BtTC - SO 40.2018
APPLICATION OF DNA MOLECULAR MARKER IN SLOW RIPENING TOMATO CONTAINING S/TVGENE
VARIETY SELECTION
Nguyen Thi Van, Nghiem Thi Huong ABSTRACT
This study uses DNA molecular marker to select elite individuals in rin gene containing F2 divided population (ripening inhibitor) which controls slow ripening characteristic, high yield, good quality in tomatoes. The experiment was conducted on a divided population derived from the hybrid tomato variety coded 159 imported from the United States by the Center for Plant Genetic Resources Conservation and Development.
Keywords: Slow ripening gene, slow ripening tomatos, rin gene.