HOI NGHj KHOA HOC C O N G NISHE SiNH HQC T O A N Q U O C 2013
XAC DINH HAM LU'CTNG BETALAIN VA MO'C DO BIEU HiEN CAC GEN THAM GIA VAO SINH T 6 N G HOP BETALAIN TREN THANH LONG
Tran Thj Oanh \en\ Ngo Van Bi^h^ Kui Un-Wa^g^ Andrew Allan^
' Vi$n Ciy in qui miin Nam
^ Phin vi$n ca 3i$n Ndng nghiep va cong ngh$ sau thu hO0.ch ' Vi^n nghien cuv Ciy trdng va Ciy tuong thuc New Zeals^d TOiW TAT
Bay gidng Ihanh long c6 mau sAc v6 va thjt qua khae nhau nhu do, dd hdng, dd nSu, vang d vo qua va mau dd, dd tim, Sm hfing, tring hdng vk tring d ihjl qua dupe xac djnh him lirpng tietalain hien dien trong vo va thjt qua va phSn dch miic dd bioi hien cua cac gen Laccase, DOD vaCYP76ADI; cac gen tham gia vSo qua trinh sinh tdng hop betalain 6v6 va thjt qua ihanh long bang ky thuat qRT-PCR, Th^h phin va h t o lu^mg t>etalain trong vo va thjt qua ciia cac gidng dianh long da dupe xac dinh bing mdy quang phd UV-VIS, k6t qua chdng td ham lupng betalain cao trong vd va thjt qud cua cdc mlu gifing thanh long co vo, thit qua mau dd. Cdc mlu vdi vd mdu dd, dd hong, dd nau va thjt qua dd lim, lim hong cd him lirpng betalain cao hon cac mdu vd v4ng vd thjt qud trdng, thanh phin chii y ^ hinh Ihdnh sic Id betalain Id betacyanin, betaxanthin hien diSn vdi mot hdm lupng rdt t h ^ ngay ca 6 cdc mlu vdi vd qud mdu vdng. Phan tich mdc dp bilu hifn ciia cac gen ma hda enzyme Laccase, DOD, va CYF76AD1, tham gia sinh tdng hpp betalain bdng qRT-PCR, di chimg td ring gen CYP76AD1 ed thS sij dyng nbir gen chi Ihj trong chpn lao giflng cho tinh trang mdu 6 vd vd thjt qud dianh long. Mdc d§ bilu hien ciia gen ndy cao d cdc gifing cd vfi vd thjt qud dfi va hoan loan ving 6 cde mlu gifing cd vd vdng, thjt qud trdng.
Tu khoa: Betalain, bieu hi^n gen, qRT-PCR, thanh long Rupt tim hdng, thanh long Vd vdng
DAT VAN Bt
Mau dirg'c xem nhu- li chi lh| v^ chat lu'g'ng it thuc phim va quy^t ^nh si/ chap nh^n cua ngU'd'i tieu dung, nhieu thi>c in c6 mdu tl/ nhien nhir cac sdn phim ti> qua nhu'ng se bj mat mau Irong qua trinh chi biin, do do c^n phai sir di^ng mau d l tai t?o miu thi/c phim. Con nginri ngay c^ng huong toi mau tv nhign, hgn che si> dung mau tong hop, vi mdu ty nhi6n IS vfl h^i hay thjim chi con c6 lai cho SLFC khfie Nhu cau nay da thuc day sy thay do! lan tren the gid'i, thj taiKrng mdu t6ng \\gp ngdy cdng suy tdn va chuyin din sang sir dung mdu tu nhign (Fletcher, 2006).
Betalain la logi sSc t6 ti/ nhiSn c6 cSu tgo la betaxanthin cho mau vang va betacyanin cho mau do, la chat ch6ng oxi hoa hilm c6 trong ti/ nhiSn. Sy phdn bo cua betalain trong tu nhiSn chl hi§n di?n gioi han doi vd'i vai loai cdy thu^c bg Caryoph^alles, cu cdi dirdng d6 (C/)er7opoc/aceae) va m$t vai lodi nam nhir ndm Fly-aganc {Amanita muscaria) (Sh'ack el al., 1993). Trong dd, cO cai du'crng do la lodi cdy trong cung d p betalain chO yeu va da dugc thuang mgi, si> dung tnjng thyc phim nhu' mdu thirc phim ir My va cdc nuoc chau Au (E162). Ngay nay, betalain dugc sir dung rgng rSi nhu' sic td ty nhifin trong cdng ngh^ thyc pham d$c biel Id mdu ir kem va yoghurt. Mflt vai bao cao nghien ciru tiin qua thuOc hg xi«mg rbng (Stintzing et aL. 2001) vd cay t h u ^ hp Amaranthaceae (Cai et a/.. 2005) nhir nguon mau thyc phim ty nhidn cd trien vgng cung d p them vao nguon betalain ti> cu cai du'crng do. Mgt nghien cCru du'yc thyc hi^n bcri Obon vd c0ng sy (2009) da chung 16 nhieu kit qua hi>a hgn bang v\dc irng dung bflt cua qua Opuntla stricta cho vdo yoghurt va nu'd'c uong. Ket qud khao sat da cho thay ring mdu cua san phim duy tri mau tim do sang sau mgt thang ton trO trong tu Ignh,
Tong hyp betalain c l n trai qua 3 enzyme xuc lac chfnh (hinh 1), Cdc enzyme c6 nhiem vu * buwc 1 va bode 3 thi chu-a du^c biet vd duvc cho la enzyme fifong ty nhu- tyrosinase [Laccase vd CyP76AD1). biln doi tynasine thanh L-3,4- dihydroxyphenylalanine (L-DOPA) va sau do thdnh dopaquinone. Dopaquinone diryc d l nghj tgo vdng ngiu nhign thdnh cyc/o-DOPA, mdc du sy hien di§n cua dopaquinone chira duvc chirng minh d dy. L-DOPA Id chdt nln ^ o buirc 2, gen sinli ting hyp dgng v6ng diryc phdn lap, ir do enzyme phdn cat mo vflng extradiol, DOPA 4.5KJioxygenase (DODA) doyc de nghj tgo ra 4,5-seco-DOPA, tgo vong ngau nhidn thanh betalamic acid. Dieu ndy da diryc chung to trong in- vitro mi acid betalamic c6 the kit tua, c6 dac mflt each nglu nhiSn vfl'i sdn pham diryc d l nghj a bircrc 3, cyc/o-DOPA, d l htnh thdnh sac to betanidin mdu do. Acid t>etalamic cung co thi duyc k l l tua vai amino add hay nhom amin khae d l hinh thdnh sic to betaxanthin mdu vang.
t/ng dung thdng thiryng nhlt cua qPCR Id phan tich mtrc dO bieu hien gen, kham phd mam bgnh vd so lifyng microRNA (Schmittgen TD e( ai, 2008). Hilu ro cac kilu bieu hien gen din tai hieu tflt hon ve cdc tiln hinh sinh hgc.
qRT-PCR hi§n Id phirong phdp chuan cho cac nghign CLPU sau v l mirc dd bilu hign gen. Ngdy nay phdn tich mi>c do bieu hign gen dang diryc nhilu nhd nghien ct>u qiian tdm nhdm khafti pha gen tham gia qui dinh kilu hinh ir smh vii Cac gen tham gia qud trinh sinh ting hyp cdc sac to anthocyanin, carolenoid, chlorophyll, flavonoid ir dc loai cdy trong dang duac nghiSn ciru vd phdn tich. Zhi Chang Zhao va cac c0ng su (2012) da phdn tich thanh phln sac t l trong mau vo dd ir qud vdi vd phSn tich mirc do bilu hien cua 4 gen tham gia vao qud trinh sinh tong hyp anthocyanin, k i t qud cho thiy ring ham liryng ciia sac t l anthocyanin va flavonol trong vfl quS vdi fl I? Ihudn vdi mi>c do bilu hign cao cua gen ma hfla enzyme UFGT va cac tdc gid cung d l nghj rang gen ma hda UFGT ddng vai tro quan trgng trong sinh ting hyp anthocyanin ir vo qua vdi, va mi>c dfl bilu hi?n ciia UFGT anh hud'ng mgnh tdt mdu cua qud vdi.
Myc dich ciia nghign cuu nay Id xdc dinh thanh phln vd ham luyng betalain cd trong thjt vd vo qud thanh long vd phdn tich mi>c dO bieu hign cua ba gen chinh tham gia vao flen trlnh sinh ting hyp betalain nhlm xdc djnh cac gen dnh hucrng quan b-png din mdu sic vo, thjt qud, ldm ca sir cho thilt k l dogn mli irng dung cho chgn tgo gidng thanh long CO hdm Ii/yng betalain cao.
HOI NGH! KHOA HOC CONG NGHE SINH HOC TOAN Q U 6 C 2013
COOH ?
• ' V 1 Hooc'^pa
HOGG N'^COOH Sstalacyanin
Red
Hlnh 1. Con du'd-ng sinh tong hon betalains.
i^ A
Budc 1, 2 va 3 Id cSc enzyme tham gia, ngirpc lai cdc budc danh ddu S duwc cho Id ngau nhien (Hatleslad ef al. 2012)
V A T U ? U V A P H U ' a N G P H A P N G H I E N CCrU V|t lif u
Bdy giong thanh long duyc su dung lam nguln vat ligu cho xdc (Snh hdm luyng betalain vd phan tich muc dfl bilu hign gen gom; gilng thanh long Ru0l trlng Binh Thudn, thanh iong Rugt do Long Dmh 1, thanh long RuOt tim hflng LDS, thanh long Ru$t 56 tim T7, thanh long Rugt trang hflng T12, thanh long Vfl vdng, Ihanh long Vo vang ddng lai B666. Day Id cdc giong/dflng thanh long c6 vo va thjt qud cfl mau sdc khae nhau duoc chgn Igc cho phdn lich hdm luyng sic l l vd mirc dfl bilu hi|n gen qui djnh tinh trang betalain.
Hlnh 2. Mdu v d va thjt qud cda giong thanh long Rudt trang Binh Thuan, Rudt do Long Djnh 1, Rudt tim hdng LDS
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Hinh 3. tJIku vd va thjt qud cua giong thanh long Ruot dd tfm T7, Rudt trdng hdng T12, Vd vdng, Vd vdng lai B666 PhiFo-ng phap nghi&n ci>u
Thu th^p mlu qua
HQI NGHI KHOA HOC CONG NGH$ SINH HOC TOAN QUOC 2013
Thu thap mau qua cho tdch chtit RNA: RNA duyc ly trich tir vo va thjt qua ciia cdc giong thanh king vdo giai dogn vfl qua vira chuyin mau (27-28 ngay sau khi hoa nir doi vd'i thanh long Rugt trang, Rugt trang hing, Rugt tim hong va Rupt do vd 56-57 ngay sau khi hoa na dfli vai nhom thanh long Vo vang). Mau qud duyc thu thap tgi trgi thuc nghigm. Vien Cdy an qud miln Nam, mlu duac n>a sach bdng nuoc clt ngay sau khi thu thdp, lam khfl bdng gily thim nuoc. Cac mdu sau dfl duoc lay phan vo vd thjt qua (khodng 5g/mdu), moi^ gilng lay 3 qud, mau sau khi cdt rai khoi quS duyc cho ngay vao dung dich nita long khodng 2-3 phut vd Idy ra gdi lai bang gidy nhom cho vdo hop nhya nho (gidy nhflm vd hgp nhya cung duac xu ly vol nita long) sau do dua ngay vdo tCi Ignh -20''C. Cdc mau sau khi duyc chuin bi xong duyc chuyin sang phong thi nghiem sinh hoc phdn tu cua Vien nghien cuu Cay trong vd cay luong thyc (New Zealand) cho ly trich RNA va thuc hign cac buac tilp theo cho phdn tii± muc do bilu hien gen.
Thu thap m l u qua cho xdc dinh ham liryng sac t l betalain: mau qua duyc thu thap tgi trgi thyc nghiem ciia Vien Cdy dn'qua miln Nam a giai doan qua chin hoan todn (30-32 ngdy sau khi hoa no, dfli vd'i thanh long ruflt trlng. mot hing vd rugt do vd 60 ngay sau khi hoa na dli vai nhom thanh long vo vang). Mau sau khi thu hoach duyc chuyen ngay tai phdng mi nghigm phdn tich cita Phan vien ca dign Nong nghigp va Cong nghe sau thu hogch cho phdn tich vd xac djnh hdm luong cua sic t l betalain co trong vo va thit qud thanh long.
Xac dfnh ham lirang betalains trong vo va thit qua thanh long: bing phuang phap do quang pho UV-VIS si> dung dung mfli tdch chiel Id ethanol 80%.
Qui trinh thuc hign: Bgt quang pho k l vd cho phep mdy tinh ghi kit qua d l lam am len it nhlt 30 phut trudc khi do. Dieu chinh mirc quang phi o cdc buac song 476, 538 vd 600nm bang each sir dung dung djch ethanol 80% idm dung moi.
Can 0,5g bflt thanh long duyc nghiin bdng nita long cho vao 2ml dung djdi ethanol 80%, sau dfl lie tren may lac trong 10 phut, dem ly ldm 9 000 vflng/phOt trong 10 phut. Hiit lly dich thanh long va tiln hdnh do bing mdy quang phfl hip thu c6 cde bufl'c song lln luyt Id 476, 538 va 600 nm
Betalain duyc tinh theo cong Ihuc. BC (mg/g) = [(A(DF)(MW)Vd/eLWd)]
Trong dfl: A: gia trj ciia buoc sdng hip thu toi da lln luyt la 538nm cho betacyanin vd 476nm cho betaxanthin; DF: do pha lodng, Vd: thi tich dung djch t}Ot kho (ml)
Wd: trgng luyng bOt thanh long dflng kho bang nilo long (g);
L: chilu ddi cua cuvette (1cm)
Vai: Cdc trgng luyng phan tir (MW) vd hg sfl dap tat phdn tir (e) eua betanln [MW: 550g/mol, E: 60.000l/(motcm) Irong nuyc) duyc dp dyng d l xdc djnh s l luyng betacyanin. Djnh luyng luong duang cua betaxanthin chinh (Bx) da duyc xdc i^nh bdng cdch dp dyng MW = 348 gam/mol vd hg so phdn tip [E: 48.0001/(mo! cm) trong HjO].
Ly trich RNA t6ng so va tinh sach RNA: RNA tong sfl duyc ly trich lu vfl vd thjt qua thanh long theo phu'ong phap CTAB (Chang et al., 1993) cd chinh sua cho phu hyp, RNA sau dfl duyc idm sgch theo qui trinh eua QIAGEN vd lam sgeh DNA bing bfl Kil DNA free treatment cua Ambion.
Thiit ki cae doan moi sO dimg eho phan tich mirc do bieu Men gen: RNA ting so sau khi duoc ly trich duyc gui
^nh chuli baza nucleotides tgi Inrdng Dai hgc Queensland (Oc). Trinh lu chull bazo nucleotides cua cdc gen Actin, DOD. Laccase va CYP76AD trgn RNA cua thanh long duoc xac dinh bing sir dung cac chuoi gen eo san trong Ngdn hdng gen quic t l ciJa B. vulgans nhu sau D0DA1 (HQ656027), B. vulgaris CYP76AD1 (HQ656023) v^ ILaccase (JQ085589), Act (HQ656028) va chiing duoc Blast tren chufli RNA thanh long nham tim ra chuoi baza nucleotides cua gen DOD, CYP76AD1 vd Laccase cua thanh long. Dua tren eac chuoi gen tim ra d l thilt ke cac mli sir dung cho qPCR.
Thiet ke cac do^n mdi cho qRT-PCR: cdc dogn mli duyc thilt bang phan mem Vector NTI ciia Invitrogen di/a vao cdc chuli bazo nucleotides trong vOng CDs (Coding DNA sequencing- ehuli DNA tham gia ma hfla) cua gen DOD, gen CYP76AD1, gen HyAcUn, vd gen Laccase, vdi nhigt dg Tm la 60*C. Cdc dogn mli cho qPCR duye thilt k l trong phgm vi90-150bp, tflt nhlt Id 120bp, trinh ty cac dogn moi duyc trinh bdy d Bang 1.
Bang 1. Cac primer diro'c sir dung cho ptian tich s y bieu hl^n gen t>ang qRT-PCR
Stt Gen Hud'ng Chuoi nucleotides (5'-> 3') 1 HvActin FonwanJ GAA GAT CCT GAG TGA GAG AGG TTA CAT GTT T
' Reveise CCT GTT CAA AAT CAA GGG CAA CAT A 2 CVPTRAn Fonirarei GAT ATA GGC AAG GCT GCTTTC ACG
Reverse AGT ACT TTA AGC ATG GGA AAT AAT C .. __
•i " n n n ' ~ FonranJ TTC CTG GCT AGA AGA AGC TCT CAA AA TGG TCC CAG CTG CGA TGA ATA A ATT CTC ATC AAC GGG CTT GG CCC ACA TTG CAG ATC CTG AG
Tong hg-p cDNA vi kiem tra cac doan mdi thiet ke
Tong hyp cDNA: sir dung bg kit ting hyp cDNA cua Invitrogen, qui trinh long hyp duyc hudnng din ctii tiet trong bO kit.
cDNA ciia cac mlu sau khi tong hc^ duac tiln hdnh nhdn doan DNA muc tidu bdng mdy nhSn phdn tu PCR, su dung cdc dogn mli chuygn bi#t d bang 1 nhlm kilm tra cdc doan moi Oiilt k l vd cDNA dd duyc tong hyp. Cac sdn pham RT-
HOI NGH! KHOA HOC CONG NGHE SINH HOC TOAN QU6C 2013
P C R (san p h a m P C R cua c D N A ) d u o c k i l m tra Iren gel agarose 1 % (w/v) v d i c h l l nhugm la S Y B R Green®, q u a n sat cac doan D N A bang Gel Doc.
Piiin-tletrmdv-G&tlt^ hien Cua geri'tbam gta vao qua tnnti tong hgp betalain Bang phuvng phap qRT-PCR T h a n h p h a n cho 1 phan i r n g q P C R (lOpI): 5pl Master mix. 3Mi d o a n m l i (primer) vd 2pl c D N A . T d t cd cde phan u n g d u y c t h y c hien sir dung Fast Start Universal S Y B R Green Master Mix (Roche) theo qui trinh d u a c k h u y i n cao b d i nha s a n x u d t Sfl lan ldp Igj cho mfli phan i r n g q R T - P C R Id 4 Idn
C h u trinh c h g y q R T - P C R : SS^C trong 5 phut, 95°C trong 15 giay, f i p theo 60°C Irong 5 giay, 72''C trong 10 gidy, lap lai 4 0 c h u ky, tiep theo la 65''C t d i SS^C kham phd d u d n g c o n g vd phdn lich hieu qua qPCR c u a mfli g e n bdng phdn t i c h d u d n g c o n g c h u a n theo p h u a n g phdp pha l o a n g hdng loat c D N A v d i cdc cdp dfl pha loang 1 0 , 100, 1 0 0 0 , 10.000 v a 100.000 lan cua c D N A ban d d u . Gen Actin d u y c s u d y n g n h u gen t h a m khao (reference g e n e ) .
S a u khi h o a n t i t c d c chu Irinh chay q R T - P C R , m u c d$ b i l u hien ciia dc gen CYP76AD, DOD va Laccase se d u o c p h d n lich b a n g p h l n m i m LightCycler@480
K t T Q U A V A T H A O L U A N
Kit qui phan tich ham lu-gng betalain tren cac glong/ddng thanh long chgn Ipc
B a y g i l n g t h a n h long duac chgn Igc cho xac djnh ham l u y n g betalain Irong vo va thit qud I h a n h long Id thanh long Ruflt trang Blnh T h u d n , thanh long Ruflt d o L o n g D i n h 1 , thanh long Ruflt tim hong LDS, thanh iong Rugt d d l i m T7, t h a n h long Rupt trang h i n g T 1 2 , thanh long V o v d n g (My), thanh long V o vdng dong lai B666. C d c giflng d u y c chon toe c o m d u sac v f l v a thit khae nhau n h u Irinh bdy d Bang 2.
Bang 2. H a u v d va thft qua ciJa bay giong thanh long d u a c c h o n loc c h o phan tich S " Tdngiong/ddng Mau vd qud iUldu thjt qua
1 Thanh long Rudt tr^ng Blnh Thudn 0 6 nau Triing 2 Thanh iong RuOt dd Long Bjnh 1 Dd sang Dd a Thanh longJamjl,tim hdng LDS Sd T i m h ^ 4 Thanh long Ru0t do tim T7 Dd nau Dd tim
5 TTianh long Ru0t trlng hdng T12 Dd tidng Trgng hong 6 Thanh long Vd vdng Vang sang Tr^ng 7 Thanh long Vd vdng ddng lai 6666 Vang sang Tr^ng Bdy giong t h a n h long tren d u o c phdn tich h a m l u o n g betalain v d t h d n h phan sac tfl cua betalain n h u betaxanthin s d c to c h o mdu v d n g v d betacyanin sac t l cho m d u do. Ket qua phan t i c h hdm l u a n g betalain d u y c trinh bdy d Bang 3 c h o thay t a l c a cdc m l u phdn tich d e u c d s y hi?n dign ciia betaxanthin v a betalacyanin, tuy nhien c d s y c h e n h I g d i I d n v l h d m l u y n g betalain giu-a cac g i l n g I h a n h long Giflng thanh long Ruflt d d Long Dinh 1 co h a m l u o n g betalain cao nhat d ca v d vd thjt qua (2,754 m g / l O g vd v a 2,633mg/10g t h j l q u a ) , k l d i n la cac giong thanh long Rugt d o tim T 7 , thanh long Rugt tlm h i n g LD5, cac m l u v d i vd cCia I h a n h long ru^t trdng vd v d cQa thanh long ruot trdng h i n g c d v 6 m d u dd n d u v a d o h i n g l u a n g i i n g , d i u c d h a m l u y n g betalain c a o . Ham l u y n g betalain rat t h i p h i u n h u khflng ed d cdc m l u c d v f l q u d m d u v d n g n h u hai dflng t h a n h long vo v a n g ; vd thit qua mau t r a n g , trang h i n g n h u thjt q u d cua g i l n g thanh long Ru$t trang B i n h T h u g n , t h a n h long RuOt t r l n g hong T 1 2 . M a t khde, betaxanthin Id h a p chat c h o sdc t l m d u vang, trong p h d n tich ndy cho t h i y cd hai m a u Id v d q u a cua giong t h a n h long v o vdng cd hdm l u y n g betaxanthin vd betalain rdt t h a p ( B a n g 3)
Bang 3. K i t qua phan tich ham l u a n g ciia betaxanthin va betalacyanin
T - , ' . 1 ' Betaxanthin Betalacyanli
Ten giong Mau r m a / I D a m i u l (mn/lna m3. (mg/lOg m i u ) (mgrtOg m l u ) ^ « ' ^ ' ^ ' " ^"'^''^^^ "
Thanh long rudt trang (Tl) Thanh long rudt dd Long Djnh 1 (T2) Thanh long mflt tim hfing LOS (T9) Thanh long ruot dd tim (T7) Thanh long ruSl tring hdng {T12) Thanh long Vd vdng ( W ) Thanh long vd vang ddng lai B666
V o q u a Thit qua V d q u a Th|l qua V d q u a Thjt qua Vdqud Thit qua V d q u a Thit qua Vfl qud Ttiit qua V o q u a Thit qud
0.595 ± 0.080 0,017 + 0.006 0,684 ± 0.023 0,671 ±0,102 0,687 ± 0,086 0.645 * 0,032 0,582 ± 0,058 0.641 + 0,042 0,598 ± 0.071 0.029 ± 0,006 0,012*0.001 0,010 ±0,001 0,016 ±0,001 0,007 10,001
-1,913±0,113 0,014 ±0,005 2,069 ±0,097 1,962 + 0,239 1,636 + 0,158 1,101 ±0,075 1.812 + 0,072 1,555 ±0,091 1,560 + 0,121 0,047 + 0,003 0,029 ± 0,003 0.005 ± 0,002 0,019 + 0,001 0,006 ± 0,002
- - 2.508 0.031 2,754 2,633 2,324 1,747 2,3S4 2,196 2,159 0,076 0,041 0.015 0,035 0,013 Phin tich mirc dd bleu Men cua cac gen tham gla vio qua trinh tSng hop betalain
T r o n g phdn l l c h m d c dO bieu hign g e n cda b a g e n DOD. CYP76AD, v d Laccase. g e n Actin d u y c s i r d y n g Id gen t h a m k h d o (reference gene hay h o u s e keeping g e n e ) G e n t h a m k h a o Id g e n ludn hign dien trong cdc m f l c d y , cdc nghien c u u v l q R T - P C R lufln c l n phai co g e n t h a m k h a o v a d u y c x e m n h u la d l i c h u n g m u c do chuan hoa eiia m R N A v d i cac
HOl NGHI KHOA HOC C O N G NGHE SINH HOC T O A N Q U O C 2013
mirc dd khde nhau giu-a cac gen khae nhau. chung cho mdl su so sanh chinh xdc cac muc dfl bieu hien gen ciia cac gen nghign cuu.
Muc do bilu hign ciia gen Laccase duac trinh bay d bilu dd 1. Ket qua bilu dd 1 eho thay vd ciJa gilng thanh long Rupt irlng hong vd vd cua Ihanh long Vd vang ruflt trlng cd mue do bilu hien gen Laccase cao nhlt. k l din Id vd eiia Idl ca ::dc gidng thanh long, thjt qud cua cac gidng thanh long cd muc bilu hien gen Laccase thip hon. Kit qua nay chirng td
"dng gen Laccase tren thanh long khong phai Id gen chuyen biet cho ung dung nghien euu chgn lao giflng thanh long cd ldm luyng belalain eao hay chgn lao gilng thanh long cd mau do, do tim bing chi thi phan lu.
L s c c a s e v s A c t i n
B i l u do 1. Mi>c dp bieu hien cua gen laccase tham gla vao sinh tdng h y p betalain
MtFc dy b i l u hien gen DOD: gen DOD cfl sy hien dien d tit ca cdc mau vd qua va Ihjl qua cua cd bay giong thanh ong, muc dd bilu hign cao d vd qua vd Uilp d thjt qua, muc dg bilu hign gen cao nhll d vd cua gilng Ihanh long Vd /dng, tuy nhien d gilng Ihanh long Vo vdng ddng lai B666, gen nay chi bilu hign d mirc dp luang duong vdi thil qua aJa gilng Ihanh long Rudl do Long Djnh 1 hay thit qua mdu trang ciia giflng thanh long Vd vang (Weu dd 2). Kit qua nay
;ho Ihly ddy chua phai Id gen chuygn bigl cho vigc ung dyng chgn tgo giong bdng chl thj phan tu vdi linh trgng mdu sac id vd thit qua tren thanh long.
D O D VS A c i t m
Bieu dd 2. M u c do b i l u hien cua gen DOO tham gla vao sinh tong h y p betalain
iflirc do b i l u hign cua gen CYP76AD' gen cytochrome P450, cd muc tuong dong cao vdi gen CYP76AD1. ma hda inzyme tham gia yao qud trinh hinh Ihdnh sdc lo betacyanin mdu do d cO cdi dudng da chirng Id cd m l i tuong quan r l l dt vdl muc dfl bilu hien sdc to dd d thanh iong. a cdc md qud mdu trlng gen ndy ed muc dfl failu hign rdi thip. Q dc nd mau vdng Ihi mire dfl bilu hien cua gen nay hodn todn khflng cd (bleu dfl 3). Kit qua nay chirng Id gen CYP76AD1 -.6 the la gen then chll duyc sir dung cho cac nghign cuu tiep theo nhdm thill k l cac doan moi irng dung cho nghien :uu chpn tgo gidng vdi tinh trgng mdu dd/vang trdn thanh long
-Jghien cuu xa hon, chiing tfli dd liln hdnh phdn lap gen ndy t!> DNA hg gen thanh long vdi cdc mfl Ihanh long mdu dd 'd vdng. Chufli DNA cua CYP76AD1 a dc md mau do khde so vdi cac md mdu vang.
3i*ud6 3.Mi>cd6bl4u C Y P v s A c t i n itf n cua gen CYP76AD1
-ham gla v4o sinh tdng ^
lyp betalain g s T
< - < - > - < , - v < ^ - < . , < , - = ^ < ^ < < ^ ^ ^ ^ ^
HQI NGH! KHOA HOC CONG NGHE SINH HOC TOAN QUOC 2 0 r 3
Ket qua Bieu do 3 cho thdy: vd ciia cac gilng Ihanh long cd mirc do bilu hien gene HyCYPADTS cao vd eao hon &iit
—quajnaii dn hi^nr^ trani] r:^|^ giAng thqnh long co vd vana va cac gilng co Ihitqua mau trang khong cd su bieu hien cua gene CYP76AD. dilu ndy cho thiy gene CYP76AD Id gene chuyen biet cho sac l l do, vdng d sdc Id vdng vd trang K^T LUAW
Ham luyng betalain cao d eac mdu mfl thanh long co mdu do, mdu dd cdng dam Ihi hdm luang betalain cdng cao vd ham luyng belalain d vd qud cao hon thit qua.
Cac gen Laccase, DOD vd CYP76AD1 tham gia vdo qud trinh tdng hyp betalain trgn thanh long. Trong dd CYP76AD1 CO tmh chuygn biet cao. ed mii'c dd bieu hign gen cao tren cac giong thanh long cd vo vd Ihit qua mau dd vd khdng cd su hien dign trgn mfl thanh long cfl mdu vang vd mdu trlng.
<3en CYP76AD1 cd the duye tilp tuc nghien ciru thiet k l cac doan mli cho nghien cuu ehgn Igo gidng bang chi thj phan
TAI Lieu THAIM K H A O
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flavonoid-3-O-glueosyllransferase (UFGT) gene regulates anthocyanin biosynthesis in litchi (Litchi chinesis Sonn.) during fruit coloration. Moi BioL Rep. 39. 6409-6415.
IDENTIFICATION OF BETALAINS AND EXPRESSION ANALYSIS OF BETALAIN GENES IN DRAGON FRUIT VARIETIES
Tran Thi Oanh Yen', Ngo Van Binh', Kui Lin-Wang ^, Andrew Allan ^
SUMMARY
Seven diagon ftuil varieties (red flesh Long Dmh I, pink purple flesh LDS, purple red flesh T7, pink white flesh T12, yellow peel and B665-hybrid with yellow peel) having different phenotypes for peel and flesh coloration such as red, brown red, pink red and yellow in fruit peel and red, pink purple, pink white and white in fruit flesh were analyzed for belalain content and gene expression.
Belalain content of tliese varieties had been determined by UV-VIS, results showed that betalain content in peel and flesh of red flesh dragon fruit Long Dinh 1 was highest Betalain contents of samples with red, pink red, brown red colour in fruit peel and red, pink purple in flesh were higher than those of samples with while and pink white flesh Expression analysis of three genes in the betalain biosynthesis pathway coding for enzymes Laccase, DOD, and CYP76AD by qRT-PCR, results suggested thai CyP76AD might play an important role in pigment determine in peel and flesh of dragon fruit. CYP76AD expression strongly influences on red color in flesh and peel ofdragon fruit and completely absent in phenotypes with yellow peel and white flesh. Deep sequencing of dragon fhiit RNA gave enough data for isolation of the key genes for betalain production - DOD, Laccase and a newly described P450 (CYP76AD1). It appears that this is the key gene for red/yellow differences in dragon fruit as its expression is absent in yellow fruit.
Gene variants are currently being isolated for this gene to allow for marker assisted selection.
Key words: Betalain, gene expression, pink purple dragon fruit, qRT-PCR, yellow dragon fruit
• Author for conespondence: Tran Thi Oanh Yen, Southem Hortkxiltural Research Insblute; Email. tranthioanhveniSqmail.com
