CVv201S162014021.pdf

KHOA HOC CONG NGHl

^ P H A T HiClM C H i T H ! PHAIM T U ADIV LIEIM QUAIM DEIM H A M L U q i M G C U R C U M I I M TROIMG CAC GIOIMG I \ I G H £

{CURCUMA LOHIEA L.) Q VIET IMAM

Nguydn VSn Khidm^ VQ Hoii Sam^ Phuang Thidn Thuong^, Tri#u Thi Unh^ Nguydn Xuin Cinh^

T6MTAT

Hgp chit curcumin trong cu nghd {Curcuma longa L) co tie dung hd trg chiia nhieu b^nh cr ngudi. Mpt sd nghidn cuu trudc day da cho th^y ham lugng curcumin trong cu nghd phu tiiudc nhi^u vao giong. Tdng cdng 24 gidng nghe da dupc xac dinh ham lugng curcumin t6ng sd bing may do quang phd. Nhdm I gdm 7 gl6ng CO ham lugng curcumin thap (0,25 - 0,62%) la N4, N6, N8, N9, NIC, N12 va N16; nhom II gom 17 gidng c6 ham lugng curcumin cao (1,80 - 7,19%) la Nl, N2, N3, N5, N7, Nil, N13, N14, N15, N17, N18, N19, N20, N21, N22, N23 va N24. Bi nhin bidt cic giong nghd co miic ham lugng curomiin khic nhau bang chi thi phin tu ADN, viing ETS ciia nhin (nrADN), gien rpsl6 cua luc lap (cpADN) da dugc tach chiet, giai trinh til. Sit dung ky thuat PCR-RPIP dl phan tich viing ETS da cho th^y cac gidng ngh& cd ham lupng curcumin th^p cho san p h ^ didn di 4 bang (0,4 kb, 0,3 kb, 0,2 kb vi 0,1 kb) va cac giong ngh^ co him lugng curcumin cao cho san pham (ii 2 bang (0,4 kb va 0,1 kb). Phan tich trinh tu gien rpsl6 da phat hi^n chi thi dac tnmg d cac gidng nghd co him lugng curcumin thap li AAA I'iTT rCATTCGTACTCATAACTCAGTTGGGTA va a gidng nghd co ham lugng curcumin cao li AAAAATmTCATTCGTACrCATAACTCAAGTTGGGTAA.

Tii khda: Curcuma longa, curcumin, ETS, FCR-RFLP, rpsl6.

i.niHrBHu

Ciy nghd {Curcuma longa L) thudc hg gimg {Zingiberaceai) cd ngudn gdc nguydn thuy tir An Dg, dupc tidng d kh^p cic nudc viing nhidt ddi chiu A ti-ong dd cd Vidt Nam OE>d Huy Bich etal., 2004). Ciy nghd la ciy gia vi thuc phfoi ddng thdi Ii ciy thudc quy. Trong y hpc cd truydn, nghd dupc str dung chira cic bdnh nhu dii ra miu, phu nu hi u uat sinh didn, chiia tri 16', sung dau, chiia da day t i tring vidm loet, ung nhpt, ghe Id, phong thip, tay chan dau nhiic, ving da, vidm gan, suy gan, thd huydt mau cam (J)d Huy Bich etal., 2004). Nghidn ciiu sinh hge hidn d^i d i cho thay curcumin trong cu nghd cd tic dung chdng oxy hda, chdng vidm, chdng ung thu, chdng ddng miu, chdng vi khuan, nam v i vi nit, Iim Iinh vdt thuong, giam colextdron, chira mdt sd cic bdnh nhu Alzhenner, dii thao dudng, vidm khdp, v.v.

(Sasaki e^a/.^002).

Ning su^t, chat lugng cu nghd {C. longa L.) phu thudc vio gidng, giai dogn sinh trudng, phit trien v i didu ki^n ngoai canh nhu khi hiu, phan bdn, dat (Hayakawa et al., 2010; Hayakawa et al., 2011a).

Vipn Dugc Ii?u

"Hpc vipn Ndng nghi?p Viel Nam

Trong sin xuat nhin bidt nhung gidng nghd chiia him lugng curcumin cao gap khd khin do cd sir gidng nhau ve cic die didm hinh thai. Xac dinh ham Iugng curcumin trong cic gidng ngh^ bing phuong phip hda hge phii dgi ddn khi thu hoach cii cin 8-9 thing sau khi trdng. Do dd ddi hdi phuong phip xic dinh nhanh, chinh xic han dd loai bd nhiing gidng nghd cd him lugng curcumin thip truAc khi trdng.

Cho ddn nay da cd mgt sd nghidn ciiu xac dinh gidng nghd cd him lugng curcumin cao bing chi thi ADN. Hayakawa v i ddng tic gia d i nhin bidt chi thi PCR-RFLP bang khudeh dai doan PCR atpF inti-on v i cit san pham PCR bing enzyra Seal ddi vdi C. longa, cho phep nhin bidt nhirng gidng nghd cd him lugng curcumin cao hayakawa et al., 2010). Dd phit hidn chi thi phin tu ADN nhan biet ddng ciy chiia him Iugng curcumin cao, Hayakawa v i ddng tic gia d i phin tich mdt sd vung microsatellite ADN luc lap (rpsl6 intion 2, petB intron 1, petB intion 2) cua cac loii trong chi Curcuma va cac gidng trong loii C.

longa thu thip tir Viet Nam, Indonesia, Nhit Ban va da xay dgng mdi lidn quan di truydn giua cic loai trong chi Curcuma v i xic dinh chi thi haplotype (A, B, C, D) trong loii C. longa tuong iing vdi nhiing ddng cay chiia h i m lugng curcumin khac nhau

NdNG NGHllP VA PHATTRIIN NONG THON - KY 2 - THANG 8/2014

KHOA HOC CONG NGHE

(Hayakawa et al., 2011a). Trong trudng hgp nghien Cliu khac, Hayakawa v i ddng tac gia da phin tich gien matK ciia ADN Iuc lap (cpDNi^, viing ETS (External Transcribed Spacer) ciia ADN nhan (nrADN) va da tim dugc bang dac trung cho cic ddng nghd trong Ioai C. longa L cd h i m Iugng curcumin thap va cap (Hayakawa et al., 2011b).

Sasaki v i ddng tie gia da phit hidn phuong phap nhan bidt phan tu ddi vdi cic loai trong chi Curcuma cd h i m lugng curcumin khac nhau trong cu nghd bang su dung phin tich he thdng dot bidn 18S rARN v i gien tmK (Sasaki et al., 2002). Nam 2009, Muiani va ddng tie gia d i xac dinh mdi hdn quan di truyen tren co sd' trinh tu doan nim giira cac gien tmS - tmfM d Iuc lap vol muc do ham lugng curcumin d- cac loai trong chi Curcuma v i cic gidng tiong loai C.

longa thu thap tii Nhat Ban, Dai Loan, Indonesia v i Viet Nam. Hg da phin tich 19 vitng trdn ADN Iuc lap bing PCR Sd lin lip lai AT ti-ong viing vi tri 216-237 bp dugc su dung de chan doan nghd cd h i m Iuong curcumin trong cii (Minani et al., 2009).

Trong nghien ciiu nay, chiing tdi thdng bio mdt sd kdt qua nghidn cuu phat hidn chi thi phin tu ADN lien quan den miic ham lupng curcumin khac nhau d cac gidng nghe Viet Nam.

2. M l TUDNG Vii PHUONG PHAP IKHEV Cliu 2.1. Ddi tugng nghidn ciiu

Ddi tugng nghidn cuu la 24 gidng nghe {C. longa L.) thu thip tir 14 tinh d Viet Nam gdm Lao Cai, Son La, Dien Bidn, Bic Can, Lang Son, Hda Binh, Quang Ninh, H i Ngi, Hung Ydn, Hai Duong, Thanh Hda, Nghd An, Quang Binh va Kien Giang. Cac gidng nghe nay do Trung tam Tai nguydn Thuc vat - Vien Khoa hge Ndng nghidp Viet Nam cung cap. Cu nghe dupc sir dung de phan tich ham lugng curcumm tdng sd, vi trdng tai n h i luoi ciia Trung tim Nghidn ciiu Cay thudc Ha Ngi - Vien Dugc lieu (Thanh Tri, H i NgO cho phan tich ADN.

2.2. Phuong phip nghidn ciiu

2.2.1. Xac dinh ham luong curcumin td'ng sd Him lugng curcumin tdng sd trong cu nghe duoc xic dinh theo phuong phap quang phd (Himesh etal., 2011). Chat chuan dugc su dung Ii curcuminoit tinh khiet (98%) do Hang Sigma Aldrich san xuat, trong dd chat curcumm I dat trdn 80%. Him luong curcumm trong cac gidng nghe duoc xac dmh trdn ca sd do mat do hap thu quang va

so sinh vdi dd tin chuan cua chiing. Mdi man xac dinh dugc do lap Iai 3 lan v i lay gia ti-i tinng bmh.

Him Iugng curcumin tdng sd dugc xic dinh bing % tiic sd mg curcumm/100 g miu cu kho tuydt ddi.

2.2.2. Tach chidt ADN tdng sd

Rd cua cic gidng nghe dugc thu tiiap va su dgng dd tich chidt DNA tdng sd tiieo Doyle & Doyle (1987). Tua ADN dugc nhiing vio axdtat aradn 3 M trong 30 giiy, nia bing cdn 70° sau dd hda tan ti-ong nuoc cat vd trung. ADN tdng sd dugc xu ly dd Ioai bd ARN bing u voi ARNza I d 37*'C trong 3 gid'. Dp tinh sach ADN duac kiem ti-a bing didn di tren gel agaroza 0,8%. Ndng dp ADN tdng sd dugc dg bing quang phd truoc khi tidn hanh PCR

2.2.3. Phin ung PCR

Vimg ETS (External Transcribed Spacer) ciia ADN nhan v i gien rpsl6 cua ADN luc lap dupc khuech dai bing phan iing PCR sii dung cap mdi die hidu nhu a bang 1. Phan iing PCR (50 pi) chiia 100- 200 ng ADN tdng sd, 1 pM mdi Ioai primer, 200 mM mdi loai dNTP, ddm PCR va 1,25 U enzym Tag polymerza. Phan iing PCR dugc tidn hinh tren miy PCR (Master Cycler hang Eppendorf) vdi chu trinh nhidt: 94''C - 5 phut; 94"C - 30 giiy; 45<'C - 30 giiy;

72°C- 90 giiy; lip lai 39 Ian tir budc 2 den budc 4;

72''C - 8 phiit.

Bang 1. Trinh tu cic cgp mdi si!r dung trong nghidn aiu ETS-F 5'-TTGCCGTCATGCGGCTATTT-3' Rpsl6-F

Trinh tu mdi ETS-R 5'-TAGAGCCCATCACAGCAATA-3'

5'-CCCCCTAGAAACGTATAGGA-3' Rpsl6-R |5'-TCGAAGTAATGTCTAAACCCA-3'

San pham PCR dugc didn di trdn gel agaroza 0,8% ti-ong dem TAE a cudng dd 100 V, b-ong 45 phiit. Sau dd gel dugc nhugm bing ethilium bromide trong thdi gian 30 phut. Bang sin pham PCR dupc kidm tra dudi den UV v i chup anh bing may chup va phan tich gel Ioai Gel Doclt^ ciia Hang UVP, Hoa K?.

San pham PCR dugc tinh chd bing Kit GeneAIl®

Expm™ theo md t i cua Hing GenAIl, Han Qudc.

Trinh tu vung ETS, rpsl6 cua cic miu dugc xic dinh trdn miy dgc trinh tu tai Vidn Cdng nghd sinh hpc - Vidn Ham lim Khoa hpc Cdng nghe Viet Nam.

2.2.4. Phan tich vimg ETS bing PCR - RFLP Vung ETS da dugc phin tich bing PCR-RFLP.

San pham PCR khudeh dai viing ETS dugc tinh chd vi cit bing enzym Hiii I dd tim cac bang dac trung 22 NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 8/2014

lidn quan ddn ham lugng curcumin. Thanh phan phan ling gdm 0,5 pg ADN (2-3 pD, ddm CutSmart lOX (2,5 pi), enzym HuA I (2 pi), nudc cat vd trimg den 25 ui. Phan ung cat dugc u d' 37°C trong 3 gia.

Kidm tra san pham cat bang chay didn di trdn agaroza 2% hi^u didn thd 70 V trong 1 gid', sau dd cic bing di^n di dugc kidm tra, xac dinh kich thudc va chup anh trong miy chup v i phin tich gel Ioai Gel Doclt^ cua Hing UVP, Hoa Ky.

2.2.5. Phin tich trinh tagienrpslSdluclap Mdi quan hd di truydn vdi miic him lugng curcumin tdng sd d i dugc phan tich trdn co sd trinh tu gien rpsl6 ciia cic miu gidng.

8 . KFT QUA VA THAO LUAN

3.1. Him lugng curcumm trong cac gidng nghd

Cac gidng nghd (N1-N24) cd ngudn gdc tir 14 tinh b nudc ta d i dugc Trung tam Tii nguyen Di truyen Thuc vat thu thip tidng trong tip doin gidng tai An Khinh, Hoai Diic, H i Ngi. Cic mau gidng d i dugc cung cap 12/2012 su dung Iim ddi tugng nghidn ciiu. Kdt q u i nghidn ciiu xic dinh him lugng curcumin tdng sd cd trong cac miu cu nghd dugc trinh biy trong bang 2. Sd lidu tu bang 2 cho thay cd 7 gidng nghd cd ham lugng curcumin thap (0,25- 0,62%) gdm N4, N6, N8, N9, NIO, N12 vi N16. Cd 17 gidng nghe cd him lugng curcumin cao (1,80 -7,19%) gdm Nl, N2, N3, N5, N7, N i l , N13, N14, N15, N17, N18, N19, N20, N21, N22, N23 va N24.

TT 1 2 3 4 5 6 7 8 9 10 11 12

Ten giong

Nl N2 N3 N4 N5 N6 N7 N8 N9 NlO N i l N12

Nguon goc

^6ng Cdng, Thanh Hoa Fan Ky, Nghe An

^ Ninh, Quang Binh if^n Chau, Son La VJuAng Thanh, DiSn Bitn Bach Thang, Bac Can Dinh Lip, Lang Son 34Thu6c, Thanh Hoa

^ho Quan, Ninh Binh Da Bac, Hoa Binh Cam Thiiy, Thanh Hoa

3a Che, Quang Ninh

Ham luong

(%)

(TB±SD) 3,03 ± 0,01 3,50 ±0,02 1,96 ±0,02 0,33 ± 0,01 4,01 ± 0,02 0,30 ±0,01 2,78 ±0,01 0,34 ± 0,01 0,39 ±0,02 0,45 ± 0,01 2,24 ± 0,02 0,62 ± 0,01

T F 13 14 15 16 17 18 19 20 21 22 23 24

Ten giong

N13 N14 N15 N16 N17 N18 NIO N20 N21 N22 N23 N24

Nguon goc

5ac Ha, Lao Cai I^ao Loc, Lang Son Fhanh Mien, Hai Duong i^hii Quoc, Kien Giang :1a Dong, Ha Noi

^ o a i Chau, Himg YSn Fhach Thanh. Thanh Hoa Fhanh Ha. Hai Duong

^ o a i Chau, Hung Yen Dioai Chau, Hung Yen Fhanh Oai, Ha Noi Fhanh pho Son La, Son La

Ham lugng

(%)

CFB±SD) 1,89 ±0,02 1,88 ±0,01 6,13 ±0,03 0,25 ±0,02 1,80 ±0,01 5,87 ±0,01 5,90 ±0,01 5,43 ± 0,01 5,67 ±0,01 4,17 ±0,02 6,40 ±0,03 7,19 ± 0,02 Ghi chii: TB: Trung binh; SD: sai sd chuan

3.2. Tich chidt ADN tdng sd

ADN tdng sd dugc tach chiet tir re ciia cic gidng nghd va kidm tia bing dien di (Hinh 1). Ngoii ra, ndng dp ADN tdng sd ciing dugc xic dinh bing phuong phap quang phd. Ham Iugng ADN tdng sd d cic mau gidng nghd cd ndng dp tir 420 - 825 ng/ul.

Chi sd A260/A280 d tat c i cac miu gidng nghd dao dpng 1,80-1,99 cho thiy cic miu ADN tdng sd da dupc loai bd ARN, cd the dugc su dung cho phin iing PCR khudeh dgi cic vung ADN dich.

Hinh 1. ADN tdng sd ctia cac miu gidng nghd (Nl- N5)

Phan tich vimg ETS bing PCR-RFLP

Hinh 2. Dien di san phdm PCR vimg ETS tk cic mSu ngh^ khic nhau (N1-N12);

N: ddi chung am, M: cic marker ADN Viing ETS cua 24 miu gidng nghd cd ngudn gdc tir 14 tinh khac nhau d nudc ta da dugc khudeh dai tao ra mdt bing duy nhat co kich thudc khoang 0,5 kb. Hinh 2 cho thay ket qua khuech dai PCR 12 mau NdNG NGHIEP VA PHAT TRIEN NONG T H 6 N - KY 2 - THANG 8/2014 ²³

KHOA HOC CONG NGHE nghd (N1-N12) cho ra san pham PCR vdi bing cd

kich thudfc khoang 0,5 kb tuong iing vai kich thudc mong mudn, trong khi dd miu ddi chiing am khong xmit hidn bang. Nhu vay vimg ETS da dupc khudeh dai thinh cdng. Kdt qua giai trinh tu cic miu gidng nghd da cho tiiay vung ETS cd dp dai khoang 0,5 kb tuong til nhu trinh tu ETS cua miu nghe co ngudn gdc tir Viet Nam va Indonesia d i dupc cdng bd bdi Hayakawa va ddng tic gia (Hayakawa etal., 2011a).

Tat ca trinh tu vimg ETS cua 24 miu gidng nghe (N1-N24) cd ham lupng curcumin tdng sd khac nhau d i dupc giai trinh ttr v i kiem tra vi tri cit ciia cac enzym giai han bing phin mem NEBcutter.

Hayakawa va ddng tic gia (2011a) d i sir dung enzym HinA cit sin pham PCR vung ETS cua cic miu gidng nghd co him luong curcumin khac nhau cd ngudn g6c tir mgt sd nuoc.

Nghien ciiu nay, da su dung enzym gidi han Hiii I cat vimg ETS cua tat ca cac miu. Tdng cdng 7 miu gidng nghe gdm N4, N6, N8, N9, NIO, N12 v i N16 co ham Iugng curcumin dao ddng 0,25-0,62% tao ra 4 bang vdi kich tiiuoc khoang 0,1 kb, 0,2 kb, 0,3 kb va 0,4 kb. Trong khi 17 miu gidng nghd cdn Iai ddu cd h i m lugng 1,80 -7,19% gdm cic m i u N l , N2, N3, N5, N7, N i l , N13, N14, N15, N17, N18, N19, N20, N21, N22, N23 va N24 tao ra 2 bing khoang 0,4 kb v i 0,1 kb (Hinh 3). Do do, su dung phuong phap PCR-RFLP cit sin pham PCR bing enzym Hiii I cd thd phan biet duoc 2 nhdm nghd cd ham lupng thdp (0,25- 0,62%) v i nhdm nghd cd ham lugng curcumin cao (1,80 -7,19%). Kdt qua nghidn ciiu ciia chiing tdi phii hgp vdi ket qua nghidn ciiu ciia Hayakawa va ddng tac gia da cdng bd (Hayakawa etal., 2011a).

M 1 2 3 4 5 6, 8 9 10 U i; 1? 14 1? l« r 19 2t» 21 N n 23 li M

Hinh 3. Cit enzym gidi han Hui I vung ETS ciia cac m i u gidn ngh$. Gidng 1-24 tuong iing vdi cac m i u gidng Nl- N24, N: (ddi chiing) ADN khdng cat enzym. M: cic marker ADN

3.3. Phan tich gien rpsl6

Viing gien rpsl6 ciia cac miu gidng nghd da dupc khudeh dai su dung mdi dac hieu. Hinh 4 cho thay ket qua khuech dai PCR mdt sd miu gidng nghd tao ra bang cd kich thudc khoang 0,27 kb tuong ung vdi kich thudc mong mudn. Nhu vay gien rpsl6 da dugc khudeh dai thanh cdng.

Trinh tu gien rpsl6 d mdt sd miu gidng nghe da dupc xic dinh. Kdt qui duoc trinh bay trong hinh 5.

Gien rpsl6 cua miu gidng N12, N16 (cd ham lupng curcumin thap) cd do dii 269 bp. Trong khi d miu gidng nghe N14, N18 (cd him lugng curcumin cao) cd do dai 271 bp. Trdn co sd trinh tu gien rpsl6 cua cic miu, d i nhan bidt dugc cic chi thi die trung trdn gien rpsl6 d cac miu nghd cd ham lugng khic nhau.

Mau gidng N12 va N16 vdi ham lugng curcumin thap cd chi thi die trung li AAATTTTTCATTCGTACTCATAA(rrCAGTTGGGT A. Trong khi miu gidng N14, N18 (ham lugng

curcumin cao) cd chi thi dac tnmg la AAAAATTTTTCATTCGTA

CTCATAACTCAAGTTGGGTAA (Bang 3). Ket qua nghien ciiu cua chiing tdi cd mdt sd sai khic so vdi nghien cuu ciia Hayakawa va ddng tac gia da cdng bd gien rpsl6 d miu gidng nghd cd ham Iugng curcumin cao cd ngudn gdc b Indonesia dai 273 bp, vdi chi thi die tnmg la AAAAAAATTnTCATTCGTACTCATAACrrCAGTT GGGTA (Hayakawa etal. 2010).

Hinh 4. Di^n di sin phdm PCR gien rpsl6 tir cic mSu gidng ngh$ khic nhau. Gidng 1-8: Mau gidng nghd

N12-N19. M: cic marker ADN 24 NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 8 / 2 0 U

KHOA HOC CdNG NGHt

Trinh til gien rpsl6 (mdu gi6ng N12)

CCCCCTAGAMCGTATAGGAGGTTTTATCCTCATACGGCTCGAACTCGA(44)CTCGAGAAATTntCATT CGTACTCATAACTCAGTTGGGTATTCTGACATAGTCCCAGGGGAATCCTTAAACATTrATTGAGCCGTCT CTAACCTCTTTTGTTrcTCTCATCCCGAGTCATFATTCTGATCCCTITCTTGAGACAATrGAAAATAGTGT TrCCTTGTTCCGGAATCCTrFATCTFTCCTTTrAAAATCATrGGGTrFAGAACATTACTTCGA (269 hp)

Trinh tu gien rpsl6 (miu giong N14)

CCCCCTAGAAACGTATAGGAGGTTTrATCCTCATACGGCTCGAACTCGA(44)CTCGA{lAAAAATnTrCA

•rFCGTACTCATAACTCAAGTrGGGTAATTCTGACATAGTCCCAGGGGAATCCTFAAACATFTATFGACXC GTCTCTAACCTCrnTGnTGTCTCATCCCGAGTCAATTATFCTGATCCCTrTCTFGAGACAATTGAAAAT AGTGnTCCTrGTrCCGGAATCCTrFATCTFFCCTrFGTAAAATCATFGGGTTFAGACATrACTrCGA (271 hp)

Trinh hr gien rpsl6 (m&u giong N16)

CCCCCTAGAAACGTATAGGAGGTnTATCCTCATACGGCTCGAACTCGA(44)CTCGAGAAATFTrTCATT CGTACTCATAACTCAGTrGGGTATTCTGACATAGTCCCAGGGGAATCCTFAAACATrFATFGAGCCGTCT CTAACCTCTTFTGTTFGTCTCATCCCGAGTCATFATFCTGATCCCTrFCTFGAGACAATTGAAAATAGTGT TTCCTrGTFCCGGAATCCTTFATdTFCCTTTGTAAAATCATTGGGTFTAGAACATFACTrCGA (269 bp)

Trinh tu gien rpsl6 (mdu giong N18)

CCCCCTAGAAACGTATAGGAGGTTFFATCCTCATACGGCTCGAACTCGA(44)CTCGAGAAAAATTFTTCA nCGTACTCATAArrCAAGTrGGGTAATFCTGACATAGTCCCAGGGGAATCCTTAAACATrFATrGAGCC GTCTCTAACCrCTnTGTrTGTCTCATCCCGAGTCAATFATFCTGATCCCTTFCTFGAGACAATTGAAAAT AGTGTrrCCTTGTTCCGGAATCCTTFATCTFFCCTFTGTAAAATCATFGGGTTFAGACATFACTrCGA

(271 bp)

Hinh 5. Trinh tf gien tpslG * mJu gi^ng nghe N12, N14, N16 v4 N18. Vi tri dim, b«o 14 trinh tif du(ic sir d«ng Mm chi thi dac trung

Bang 3. N h ^ hi^t chi thj dgc tnmg tr&n gien rpslS cho cac miu gi6ng nghfe Mau giong nghg

N12

N18

Chi thi dac tnmg AAATTnTCATTCGTACTCATAACTCAGTTGGGTA AAAAATFFrrCATFCGTACTCATAACTCAAGTFGGGTAA AAATrTTTCATTCGTACTCATAACTCAGTrGGGTA AAAAATrTTTCATFCGTACTCATAACTCAAGTTGGGTAA

Chi thi phan to ADN 14 doan ADN hoac hinh tir hoc trong cSy thudc (Kalpana et al., 20O4). Trong gien nam 6*n NSF cua nhan hoac ty th^, lap th6 h«n " 8 " ™ cuu nay, chiing toi da nhan hiit dupc 7 gidng k^t vM gien hoac tinh hang (Srivatsava etal., 2009). "K^t co ham luong curcumm tdng sd th^p va 17 Chi thi phan to ADN cd do chmh xac va Un cay cao Si™K nghS cd h t a lupng curcumin tdng sd cao bing hon so vdi chi thi hmh thai va hda hpc hoi vi thdng <^W « ^ N qua phan dch vimg ETS bang ley thujt tin di huyin la duy nhft khdng phu thudc vao todi, PCR-RFLP. Chung tdi cQng nhan dang dupc chi thi c4c diiu kien smh ly v4 mdi bvong. Chi thi phan to ''So tnmg h4n gien rpsl6 dai didn cho c4c gidng cd ADN dupc sir dung d i nhan bidt lo4i, phan hch mdi ^ i m lupng curcumm cao va thip. Kdt qu4 nghidn lidn quan di huydn giiia cic lo4i, giiia cac gidng "^ * « ^^V ™ * d su dung cic chi thi phan to ADN hong lo4i, c i c c i thd tiT)ng mdt loii, mdi quan hd di "^y <•* nl"'" ^^i <:*« gidng nghd cd miic h4m lupng hniydn voi him lupng cic hoat chat co ho?t tinh smh curcumm kh4c nhau.

N O N G NGHlfP VA PHAT TRIEN NdNG THON - KY 2 - THANG 8/2014 ^^

KHOA HOC CONG NGHl

4 . KET LUAN

Phan tich quang phd x i c dinh h i m lupng curcumin trong 24 gidng nghd cd ngudn gdc tir 14 tinh b nudc ta d i cho thay: 7 gidng gdm N4, N6, N8, N9, NIO, N12, N16 cd miic ham lugng curcumin thap (0,25-0,62%) v i 17 gidng gdm Nl, N2, N3, N5, N7, N i l , N13, N14, N15, N17, N18, NIO, N20, N21, N22, N23, N24 cd miic ham luong curcumin cao (1,80- 7,19%).

Phin tich vimg ETS bing PCR- RFLP da cho thay 7 gidng nghd cd h i m lupng curcumin thap (0,25-0,62%) tao ra 4 bang vdi kich thudc khoang 0,1 kb, 0,2 kb, 0,3 kb va 0,4 kb, ti-ong khi 17 gidng nghe cd him lugng curcumin cao tao ra 2 bing vdi kich thuoc khoang 0,4 kb va 0,1 kb.

Phin tich trinh tu viing gien rpsl6 d i cho thay chi thi dac tnmg d cac gidng nghe cd ham Iuong

curcumin thap la AAAl'lTTTCATTCGTACTCATAACrrCAGTrGGGT

A vi d cac gidng nghd cd ham lugng curcumin cao la AAAAATnTTi.Vi l>"' ; \ ; • •, i-_A. k A A G T T G GGTAA.

UJICAMON

Chiing tdi chan thinh cim on Ban Giam ddc Trung tim Tii nguyen Di truyen Thuc vat - Vien Khoa hoc Nong nghiep Viet Nam da cung cip cac miu gidng nghe. Xin chan thinh cam on Ban giam dd'c Trung tam Nghien cm cay thudc va Lanh dao Vien Duoc lieu - Bo Yte da giup do va tai tra kinh phi cho nghien cuu niy.

Till UEU THAM KHAO

1. Doyle J. J. and Doyle J. L. (1987). A rapid ADN isolation procedure for small quantities of fresh leaf tissue. PhytochemicalBulletin 19:11-15.

2. Dd Huy Bich v i ddng tac gia (2004). Ciy thudc va dgng vat lam thudc d Viet Nam, I: 894-895.

Nxb. Khoa hpc va Ky thuit. Ha Npi.

3. Hayakawa H., Kobayashi T , Minamiya Y., Ito K., Miyazaki A., Fukuda T , Yamamoto Y., (2010).

Molecular Identification of Turmeric {Curcuma

longa, Zwgibercea^ with a High Curcumin Content Joumal of Japanese Botany 85, (5): 263-269.

4. HayakawaH-.MinaniyaY., Ito K., Yamamoto Y., Fukuda T. (2011b). Difference of Curcumin content ui Curcuma longa L. (Zingiberaceai) caused by hybridization with other Curcuma species.

American Joumal of Plant Sciences, (2): 111-119 5. Hayakawa H., Kobayashi T., Minaniya Y., Ito K., Miyazaki A , Fukuda T , Yamamoto Y.

(2011a). Development of a molecular marker to identify a candidate line of turmeric {Curcuma longa L.) with a high Curcumin content. American Journal of Plant Sciences, (2): 15-26.

6. Hmiesh S., Sharan P. S., Mishra K., Govind N., Singh A. K (2011). Qualitative and quantitative profile of curcumin from etanolic extract of Curcuma longa. International research Joumal of Pharmacy, 2(4):180-184.

7. Kalpana J., Warude P. C. and Bhushan P.

(2004). Molecular markers in herbal drug technology. Current Science. 87(2): 159-165.

8. Minami M., Nishio K., Ajioka Y., Kyushima H., Shigeki K, Kinjo K, Yamada K., Nagai M., Satoh K. and Sakurai Y. (2009). Identification of Curcuma plants and Curcumm content level by DNA Polymorphisms in the tmS-tmfM intergenic Spacer in Chloroplast DNA, Joumal of Natural Medicines, 63, (l):75-79.

9. Sasaki Y., Fushimi H., Cao H., Cai S. Q. and Komatsu K. (2002). Sequence Analysis of Chinese and Japanese Curcuma Drugs on the IBS rRNA Gene and tmK Gene and the Application of Amplification- Refi-actory Mutation System Analysis for Their Authentication. Biological & Pharmaceutical Bulletin,!^, (12):1593-1599.

10. Snvatsava S. and Nidhi M. (2009). Genetic Markers - A Cutting Edge Technology in Herbal Drug Research. Joumal of Chemical and Phamiaceutical Research, 1(1): 1-18.

26 NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 8/2014

KHOA HOC CdNG NGH$

DEVELOPMENT OF DNA MOLECULAR MARKERS RELATED TO VARIOUS CURCUMIN CONTENTLEVELSOFTORMERIC GENOTYPES {CURCUMALONGAL.) IN VIETNAM

Nguyen Van Khiem', Vu Hoai Sam^ Phuong Thien Thuong', Trieu Thi linh^, Nguyen 3&ian Canh^

Summary

Curcumin, which is traditionally known to have effects on various types of diseases in humans, is found in Curcuma longa L Previous reports indicated that the curcumin content varies between the different lines of this species, depending on other Actors such as genotypes. Total curcumin content of 24 genotypes was determined using spectrometer. Group I was included 7 genotypes with low total curcumin content (0.25 - 0.62%) such as N4, N6, N8, N9, NIO, N12 and N16; group II was included 17 remaining genotypes wifli h i ^ total curcumin content (1.80 -7.19%) such as N1J^2, N3, N5, N7, Nil, N13, N14, N15, N17, N18, N19, N20, N21, N22, N23 and N24. In order to develop DNA molecular markers related to various curcumm content levels of candidate geno^fpes of Curcuma longa L originated from 14 difference provinces in Vietnam, ETS (Extemal Transcribed Spacer) region in nuclear DNA (nrDN^ and rpsl6 gene in chloroplasts (cpDN^

were amplified and analyzed. PCR-RFLP method was used to analyze ETS region. The results showed that low curcumm content containing genotypes gave 4 bands (0.4 kb, 0.3 kb, 0.2 kb, 0.1 kb) and high curcumin content containing genotypes gave 2 bands (0.4 kb, 0.1 kb). Anatfzmg of sequences of rpsl6 gene investigated specific markers in low curcumm containing genotypes such as AAATlTlTCATTCGTACTCATAACTCAGTrGGGTA and in high curcumm having geno^fpes such as AAAAATTTTTCATTCGTACTCATAACrCAAGTTGGGTAA. These markers could be used for identification of turmeric genotypes containing low and high curcumin content levels, respectively.

Keywords: Curcuma longa, curcumin, ETS, PCR-RFLP, rpsl6.

Ngudi phin bidn: PGS.TS. Phgm Xuin Hdi Ngiy nhin bii: 12/5/2014

Ngiy thdng qua phin bidn: 12/6/2014 Ngiy duy^t ding: 19/6/2014

JdNG NGHIEP VA PHAT TRI^N N 6 N G THON - ICV" 2 - THANG 8/2014 '^'^

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