Kỹ thuật di truyền trong công nghệ chọn tạo giống hoa

Nội dung text: Kỹ thuật di truyền trong công nghệ chọn tạo giống hoa

1. Tgp chi Cdng nghi Sinh hgc 7(4): 397-410, 2009 BAI TONG QUAN KY THUAT DI TRUYEN TRONG CONG NGHE CHON TAO GIONG HOA Duong Tan Nhut', Bui Van Thl Vinh^ Trdn Trong Tuan' Viin Sinh hgc Tdy Nguyen ^Truang Dgi hgc Ky thudt Cdng nghi thdnh phd Hd Chi Minh TOM TAT Nganh cdng nghiep frong hoa fren the gidi hien dang phat frien rat manh me, nhu cau tieu thu hoa la rat ldm, vi the van de nghien cim chon tao gidng hoa mdi, nang suat cao nham dap img nhu cau hien nay la van de cap thiet. Ky thuat di truyen la phuong each quan trpng trong viec md rpng ngudn gen nham thuc day viec tao ra cac gidng cay hoa thuang mai mdi. Su thuong mai hda ddi vdi cac loai hoa dugc bien ddi di tmyen hien nay chi ban che d cac loai hoa cam chudng cd mau sac khac la. Nhimg kien thuc nay dang dupc img dung de dat dugc su hieu biet toan dien hon d pham vi rpng hon ddi vdi nhieu loai hoa khac nham muc tieu hudng vao su bien ddi mau sac d cac loai hoa do. Cac tinh trang khac cung da thu hut duoc su quan tam cua cac nha khoa hpc bao gdm huong thom cua hoa, hinh dang cua cay va hoa, qua trinh lao hda (tan) ca khi hoa d fren cay cflng nhu sau khi thu hoach va tinh khang benh cua hoa. Nhflng ilng dung cua ky thuat di tmyen tten nhflng dac tinh mdi ttong su da dang da cd dua vao kha nang chuyen gen thuc vat va dieu nay dang dugc tiep tuc md rpng nghien cim vdi tdc dp nhatih. Tie khoa: Gen, hoa, ky thuat di truyen, mdu sac, Torenia SU THAY DOI MAU SAC HOA Cac anthocyanin c6 mau sac dau tien, anthocyanidin 3-0-glucoside, c6 the dugc bo sung Sinh tong hop flavonoid va man sac hoa them dudng, cac acid beo, cac acid tham va cac nhom methyl. Co su khac biet ca ve tinh chuyen Mau sac hoa chu yeu phu thuoc vao ba loai sac biet giong, loai trong pham vi cua su bien doi va to: flavonoid, carotenoid va betalain. Betalain chiem loai nhom acyl va glycosyl gan vao loi pban tir so lugng it nhat frong ba sac to tren va tao ra cac anthocyanidin. Tuy nhien, mau sac co the nhan mau sac khac nhau nhu mau nga, vang, cam, do va biet dugc cuoi cimg cua mgt hoa thudng la sy ket tim (Forkmann, 1991). Carotenoid la nhiing hgp ciia mgt so cac nhan to bao gom kieu tich liiy tetraterpenoid 40C c6 the boa tan trong lipid va khu anthocyanin, sy bien doi tbanh phan tir tni frong cac the hat dong thdi tao ra phan ldn sac anthocyanidin, pH khong bao va dong sac tl. Mli vang trong mgt so loai boa (Forkmann, 1991). mgt nhan to deu dugc dieu boa bdi mgt sl gen va Carotenoid, cung vai anthocyanin do bay do tuoi, nhieu gen trong so chiing da dugc tao dong va xac ciing tao ra mau cam/do, mau dong thiec hay nau dinh. dugc nhin thay a cac loai hoa nhu hoa bong va hoa ciic. Flavonoid la nhom thong dyng nhat frong ba Su bien thocyanin loai sac to gdp phan tao ra mgt dai cac mau sac tit Anthocyanin co the xuat hien nhu 3-0- vang den do va xanh. Chung la cac hgp chat boa tan monoside, 3-Obioside va 3-0-tiioside ciing nhu la frong nudc va co d rat nhieu loai thye vat. Cac phan 3,5-0-diglycoside va 3,7-0-diglycoside kit hgp vai tir flavonoid dong gdp chinh vao sy tao nen mau sac cac dudng glucose, galactose, rhamnose, arabinose cua hoa la anthocyanin, do la tat ca cac O-glycoside va xylose (Strack, Wray, 1993). Cl mgt vai loai nhu (Stafford, 1990) va thudng khu tni trong cac khong hoa hong, anthocyanidin 3-0-glucoside thudng bi bao cua cac te bao bieu bi canh boa. Cac pban til dudng boa a vi tri 5- bdi UDP (glucose anthocyanin flavonoid la chat chuyen boa thil cap cua con dudng phenylpropanoid. Con dudng flavonoid tao ra cac 5-0-glucosylfransferase-5GT) dl tag ra anthocyanidin anthocyanin c6 mau sac dau tien, anthocyanidin 3-0- 3,5-0-diglucoside. Cac loai nhu Petunia va cay boa glucoside, dugc bao ton d hau het cac loai thuc vat. buam, chila mgt UDP rhanmose la anthocyanidin 3- O-glucoside rbamnosyltransferase gdn mgt nhom 397
2. Tgp chi Cdng nghe Sinh hgc 7(4): 397-410, 2009 BAI TONG QUAN KY THUAT DI TRUYEN TRONG CONG NGHE CHON TAO GIONG HOA Duong Tan Nhut', Bui Van Thl Vinh^ Tran Trong Tudn' Viin Sinh hgc Tdy Nguyen Truang Dgi hgc Ky thudt Cdng nghi thdnh phd Hd Chi Minh TOM TAT Nganh cdng nghiep trdng hoa tren the gidi hien dang phat triSn rit manh me, nhu cau tieu thu hoa la rat ldn, vi the van de nghien cim chpn tao gidng hoa mdi, nang suat cao nham dap ilng nhu cau hien nay la van de cap thiet. Ky thuat di tmyen la phuong each quan ttpng trong viec md rpng ngudn gen nham thuc day viec tao ra cac gidng cay hoa thuang mai mdi. Su thuang mai hda ddi vdi cac loai hoa dugc bien doi di tmyen hien nay chi ban che d cac loai hoa cam chudng cd mau sac khac la. Nhflng kien thirc nay dang dupc ung dung de dat dugc su hieu biet toan dien hem d pham vi rdng hon ddi vdi nhieu loai hoa khac nham muc tieu hudng vao su bien ddi mau sac d cac loai hoa do. Cac tinh ttang khac cflng da thu hut dugc su quan tam cua cac nha khoa hpc bao gdm huong thom cua hoa, hinh dang cua cay va hoa, qua trinh lao hda (tan) ca khi hoa d tren cay cflng nhu sau khi thu hoach va tinh khang benh cua hoa. Nhung ung dung cua ky thuat di tmyen tten nhflng dac tinh mdi ttong sir da dang da cd dua vao kha nang chuyen gen thuc vat va dieu nay dang dupc tiep tuc md rpng nghien cihi vdi toe dp nhanh. Tif khoa: Gen, hoa, ky thuat di truyen, mdu sac, Torenia SU THAY DOI MAU SAC HOA Cac anthocyanin c6 mau sac dau tien, anthocyanidin 3-0-glucoside, co the dugc bo sung Sinh tong hop flavonoid va man sac hoa them dudng, cac acid beo, cac acid tham va cac nhom methyl. Co sy khac biet ca ve tinh chuyen Mau sac hoa chii yeu phu thuoc vao ba loai sac biet giong, loai trong pbam vi cua sy bien doi va to: flavonoid, carotenoid va betalain. Betalain chiem loai nb6m acyl va glycosyl gan vao loi phan tu so lugng it nhat trong ba sac to tren va tao ra cac anthocyanidin. Tuy nhien, mau sac c6 the nhan mau sac khac nhau nhu mau nga, vang, cam, do va biet dugc cuoi cimg cila mgt hoa thudng la su ket tim (Forkmann, 1991). Carotenoid la nhiing hgp cua mgt so cac nhan to bao gom kieu tich liiy tetraterpenoid 40C c6 the hoa tan trong lipid va khu anthocyanin, sy bien doi thanh pban til tni trong cac the hat dong thai tao ra phan lan sac anthocyanidin, pH khong bao va dong sac to. Moi vang trong mgt so loai hoa (Forkmann, 1991). mgt nhan to deu dugc dieu boa bai mgt so gen va Carotenoid, cung vai anthocyanin do hay do tuai. nhieu gen trong so chiing da dugc tao dong va xac Cling tao ra mau cam/do, mau dong thiec hay nau dinh. dugc nhin thay a cac loai boa nhu hoa hong va boa ciic. Flavonoid la nhom thong dung nhat trong ba Su bien rhocyanin loai sac to gop phan tao ra mgt dai cac mau sac tir Anthocyanin co the xuat hien nhu 3-0- vang den do va xanh. Chiing la cac hgp chat boa tan monoside, 3-Obioside va 3-0-trioside ciing nhu la frong nuac va co d rat nhieu loai thye vat. Cac phan 3,5-0-diglycoside va 3,7-0-diglycoside kit hgp vai tit flavonoid dong gop chinh vao sy tao nen mau sac cac dudng glucose, galactose, rhamnose, arabinose cua hoa la anthocyanin, do la tat ca cac O-glycoside va xylose (Strack, Wray, 1993). Cl mgt vai loai nhu (Stafford, 1990) va thudng khu tru frong cac khong boa bong, anthocyanidin 3-0-glucoside thudng bi bao cua cac te bao bieu bi canh boa. Cac pban tir duang hoa a vi tri 5- bdi UDP (glucose anthocyanin flavonoid la chat chuyen boa thii cap cua con dudng phenylpropanoid. Con dudng flavonoid tao ra cac 5-0-glucosylfransferase-5GT) dl tao ra anthocyanidin anthocyanin c6 mau sac dau tien, anthocyanidin 3-0- 3,5-0-diglucoside. Cac loai nhu Petunia va cay hoa glucoside, dugc bao ton a hau bet cac lo^i thye vat. buam, chiia mgt UDP rhamnose la anthocyanidin 3- 0-glucoside rhamnosylfransferase gdn mgt nhom 397
3. Duang Tan Nhyt et al rhanmose vao vi tri gan glucose cua phan tir se khong co tac dgng dang ke nao den mau sac hoa. anthocyanin de tao ra anthocyanidin 3-0-mtinoside. Sy methyl hda d vi tri 3' va 5' cua vong Mgt UDP-glucose c6 hoat tinb anthocyanin 3'- anthocyanidin glycoside ciing c6 the xay ra. Sy glucosyltransferase dugc phat hien frong cay long methyl boa cua cac bat sac to co nguon goc tir dam chuyen nh6m glucose mgt each cbuyen biet den cyanidin dan den su tao thanh peonidin. Sy methyl vi tri 3' cua delphinidin 3,5-0-diglucoside. Khi b6a d vi tri 3' cua cac sac to c6 nguon goc tir cDNA ma boa cho enzyme dong bieu hien vdi mgt delphinidin ddn den den sy tao thanh petunidin; cDNA 5GT cua cay Torenia a mgt dong Petunia ma trong khi do, sy methyl b6a a cac vi tri 3' va 5' dan dong nay tich ty cac sac to delphinidin 3-0- den sy tao thanh malvidin. Them vao d6, su methyl glucoside mgt each binh thudng, delphinidin 3,5,3'- boa malvidin a cac vi tri 5-0 va 7-0 de tao O- triglucoside da dugc tong hgp. Tuy nhien, tac capensinin (5-0-methyl malvidin) va 5,7-di-O- dgng delphinidin 3,5,3'-0-friglucoside cua no len methyl malvidincan ciing xay ra a mgt vai loai thuc mau sac hoa khong the quan sat dugc do ham lugng vat (Harbome, 1967). Mgt nhom lan cac gen ma hoa thap (Fukuchi-Mizutani et al, 2003). Nhieu cho methyltransferase da dugc pban lap (Ibrahim, anthocyanidin glycoside ton tai a dang cac dan xuat Muzac, 2000); tuy nhien, cac gen dac trung dan den cua acylated (Many anthocyanidin glycoside exist in sy bien doi anthocyanin da dugc tao dong tir cay the form of acylated derivatives). Cac nhom acyl bo Petunia va cay Torenia (Quattrochio et al, 1993). sung anthocyanidin glycoside co the dugc chia ra thanh hai nhdm chinh dya vao cau tnic cua chimg. Cac dong sac to Cac nhom acyl beo (aliphatic) bao gom malonic acid Flavonol va flavone la cac dong sac to thong hay succinic acid va nhom acyl thom (aromatic) bao dyng tao nen sy on dinb va gop pban vao sy b6a gom hydroxy ciimamic acid nhu p-coumaric acid, xanh cac anthocyanin bang each hinh thanh cac phirc caffeic acid va feralic acid. Cac aromatic acyl hgp vai chung (Goto, Kondo, 1991). Flavonol dugc fransferase cDNA xuc tac su chuyen mgt nhom acyl tao ra tir dihydroflavonol do boat dgng cua flavonol tham den vi fri 3 hay 5-0-glucase ciia anthocyanin synthase (FLS). Cac gen ma boa FLS da dugc tao da dugc pban lap tir cay tia to (Yonekura-Sakakibara ddng tir nhieu loai thuc vat khac nhau. Flavone dugc et al, 2000), cay long ddm (Fujiwara et al, 1998) va tong hop tir flavanone nhd flavone synthase (FNS). cay Torenia. Mgt gen cua cay Petunia ma boa Co den bai loai FNS, mgt loai dioxygenase (FNSI) aromatic acyl transferase xiic tac sy cbuyen mgt va mgt loai cytochrome P-450 (FNSII). Gen FNSII nhom thom den 3-ratinoside ciia anthocyanin 3-0- da dugc tao dong tit cay Torenia (Akashi et al, glucoside da dugc phan lap. Cac enzyme nay chuyen 1999), hoa dong tien (Martens, Forkmann, 1999) va hydroxycin-namoic acid (p-coumaric acid hay cay tia to (Kitada et al, 2001). Mgt gen FNSI a cay cinnamic acid) den cac vi fri glucose cbuyen biet cua mui tay ciing da dugc tao dong (Martens et al, cac anthocyanin. Sy acyl boa chat tham gop phan tao 2003). nen sy boa xanh va su on dinh cac anthocyanin do do tao nSn mau sac hoa (Honda, Saito, 2002). Gia tri pH khong bao Gan day, mgt dong cDNA ma boa cho malonyl Gia tii pH khong bao, bau het thuang dugc duy Co A (anthocyanidin 3-0-glucoside-6"-0-malonyl- tri d dang acid yeu, quyet dinh su on dinh va mau sdc fransferase) da dugc tao dong tir cac canh hoa thugc anthocyanin. Mac du pH cao, ban (pH trang tinh) dugc, sau do cho bieu hien frong mgt dong Petunia thudng lam cho cac mau sac boa hoa xanh ban, tich liiy cyanidin 3-glucoside. Mac du co den 60% iihung cac anthocyanin se cang kem on dinh d pH anthocyanin da bi malonate boa nhung khong co su cang cao va phai dugc on dinh vai nhilu ban mgt thay doi mau sac dang ke nao dugc ghi nhan (Suzuki nhdm glycosyl va acyl tham (Honda, Saito, 2002). et al, 2002). Kit qua nay khong dang ngae nhien vi Sy kiem soat di trayen cua pH khong bao d canh boa sy acyl boa chat beo khong lam thay doi pho da dugc xac dinh d cay Petunia va cay bim bim hoa anthocyanin nhimg anh huang den su on dinh va sy tia. Chi CO gen cau tnic boat boa sy dilu hoa pH boa tan cua cac anthocyanin. Ngoai ra, mgt malonyl khong bao de ma boa mgt Na^H"^ antiporter (mau CoA (anthocyanidin 5-0-glucoside-6"-0- tia) d nhiing boa na buoi sang (Fukada-Tanaka et al, alonylfransferase) cDNA da dugc tao dong tir boa 2000). Gen bieu hien hoat dgng a mile do cao tai ciia cay xo do {Salvia splendens) (Suzuki,e/ al, thdi diem ngay trade khi hoa nd, ding thai c6 sy gia 2001). Su bieu hien cua no trong thye vat cho din tang pH tii 6,5 din 7,5 va sy thay dii mau sdc til tim nay van chua dugc bao cao nhung ngudi ta nghT rang sang xanh. Cac chdt dong phan da dugc phan lap tir 398
4. Tgp chi Cdng nghe Sinh ngc 7(4): 397-410, 2009 . cay Petunia, Torenia va Nierembergia (Yamaguchi lugng anthocyanin trong boa cua cay thulc la (Lloyd et al, 2001) nhung ngudi ta van chua biet chirc nang et al, 1992). Sy bilu hien cila cac gen gilng nhau ciia chiing trong ca the song. pH khong bao thudng cung ddn din sy gia tang ham lugng anthocyanin cao ban 6 cac te bao bieu bi nai ma cac anthocyanin trong cac mo hoa va mo sinb duang, bao gom la cua tich liiy (Yoshida et al, 1995). Dieu nay cho thay cac cay Petunia chuyin gen. Cac la nay se c6 mau rang, tinh ngi can bang te bao chat co kha nang la tia do sy tich tu ciia anthocyanin, va co the dac tm'ng mgt yeu to quyet dinb trang thai khong on dinh cua cho mgt loai cay mang tinh trang mdi co gia tri pH khong bao va co the gay ra bat lgi doi vai sy thuang mai (Bradley et al, 1998). Tuy nhien, cac nl song cua te bao. Sy gia tang cua pH khong bao nha lyc tuang ty dl lam tang cudng qua trinh sinh tong mgt antiporter gen trong cac thye vat chuyen gen van hgp anthocyanin a cay cam chudng va hoa hong sir chua dugc bao cao. dung cac gen tuong ty deu that bai trong viec tao ra cac cay hoa co sy gia tang mgt each dang ke qua Mgt so lugng lan cac dong cDNA ma b6a cho trinh sinh tong hgp anthocyanin hay dan den sy giam cac enzyme co tiong cac chu tiinh anthocyanin va qua trinh sinh tong hgp anthocyanin. Cac ket qua cho flavonoid da dugc tao dong va co the sir dyng trong thay sy ban che frong viec iing dyng rgng rai chien cac ky thuat phan tit de dieu khien mau sac boa. Cac luge nay de tao ra cac tinh trang la. Cac tuong tac da chien luge sir dyng antisense thudng dugc ap dyng ylu to giira cac nhan to phien ma c6 the gay ra cac de dieu khien ngugc doi vdi sy bieu hien gen d thye bau qua khong ludng trade dugc nhu sy canh tranh vat. Gan day, mgt dang lai ciia hai RNAi da dugc giUa cac nban to vdi nhau de ket hgp vai cac nban to phat frien nhu mgt cong cy manh me cho sy dieu hoa di trayen. am doi vol mgt frinh ty dich a thuc vat (Wang, Waterhouse, 2001). Trong mgt vai trudng hgp, tan NGHIEN CUU BIEN DOI MAU SAC HOA so xuat hien nhiing bien doi kieu hinh la lan ban 50% va kieu hinh on dinh ban kieu hinh thu dugc Cac nghien cihi tao ra hoa mau trang khi sir dyng antisense bay nhan to dong kim ham (Mizutani et al, 2003). Sy dieu khien ngugc cua gen cau tnic sinh tong hgp anthocyanin da dugc ghi nhan a nhieu loai thye Cac nhan to dieu hoa ciia cac gen flavonoid va vat. Vi hien tugng lai va chgn giong dot bien ciing ling dung ciia chiing de bien doi mau sac hoa dan den su phat frien cua cac giong hoa trang; cac Sy bieu bien cua cac gen cau tnic bay cac giong hoa frang la ket qua cua cac thi nghiem mo enzyme ciia qua trinb sinh tong hgp flavonoid d hoa, hinh sir dung ky thuat chuyen gen thuong dat tien la va cay con cila nhieu loai thye vat nhu Petunia, ban chgn giong truyen thong. Vi vay, sil dung cong boa mom cho, hoa dong tien, cam chuang, hoa hong, cu phan tit de chgn mgt giong trang chi co the dugc cat tudng, Arabidopsis, nho va cay tia to deu dugc thuang mai hda khi mau sac hoa mong muon dugc dieu khien ve mat khong gian (Moi et al, 1998). Hai tao ra dong thai vdi viec khong lam mat di bat cii hg gen, dang xoan vong xoan ca ban va cac nhan to dac diem co lgi nao khac. Dac biet, khi cac cay dich phien ma kieu Myb, chu yeu dieu khien su bieu hien la cay bat thy bay Cac cay tiong chuyen gen dugc tao cua cac gen cau tnic (Springob et al, 2003). Sy xuat ra CO kieu mau sac la, cac thao tac ky thuat di trayen hien cua protein WD40 trong qua trinh dieu boa CO the dugc sit dung ket hgp vdi phuang phap chgn Cling rat pho bien. Chung da dugc xac dinh ro d cay giong trayen thong. • hoa mom cho. Petunia, Arabidopsis, bap va cay tia to Cac loai hoa mau trang co the dugc tao ra tit (Springob et al, 2003). Cac gen dieu hoa ket hgp vai cac loai hoa san sinh anthocyanin bang each dieu chu trinh anthocyanin dugc bao ton ve mat chirc khien giam qua trinh bieu hien cua mgt trong sl nang trong cac loai thye vat nhung giira chiing ciing nhieu gen dieu boa hay gen cau tnic. Su lam giam CO su khac biet ve tap hgp cac gen dich, dieu nay it qua trinh sinh tong hgp anthocyanin da dugc thye ra CO the ly giai dugc tinb da dang cbuyen biet a mgt hien thanh cong d cay Petunia (Van der Krol et al, vai loai. 1988), hoa ding tiln (Elomaa et al, 1993), hoa ciic Mgt su gia tang ham lugng anthocyanin da dugc (Courtney-Gutterson et al, 1994), boa hlng ghi nhan thong qua sy bieu hien -vugt nguang cua (Gutterson, 1995), hoa cam chuang (Gutterson, cac gen ma boa cac nhan to phien ma. Vi du, Lc 1995), Lisianthus (Kato et al, 2001) va Torenia allele gen (bHLH) a bap duai sy kiem soat cua mgt (Mizutani et al, 2003). Gdn day, Nishihara va ding promoter (CaMV35S) dan den sy gia tang ham tac gia (2003) da sit dung mgt antisense iic chi gen 39S
5. Duang Tan Nhyt et al. CHS cua cay long ddm mau xanh (Gentian triflora) gen F3'5'H a cay Petunia va Lisianthus co tac va thu dugc cac cay long ddm chuyin gen mau dgng tryc tilp din sy tao ra mau sac xanh d hoa cua xanh xam. Cong ty Hokko Chemical Industry (Nhat cay Petunia va cay thulc la (Shimada et al, 1999). Ban) Cling da tao ra cay hoa anh thao chuyen gen Viec dua vao gen F3 '5 'H dugc tach chiet tir cac vai cac miic do dilu khiin giam ciia CHS. Cac mau trang boa cua cay canterbury dan den ket qua la cac sac hoa thu nhan dugc la frdng, do, hlng va mgt boa CO ty le delphinidin lan ban (99%i delphinidin) mau phoi trgn cua do va trang. so vdi khi cac gen F3'5'H cua cay petunia hay Lisianthus dugc dua vao (Okinaka et al, 2003). Gen CHS la gen dich thong dung nhdt cho sy Dilu nay xay ra co the do boat tinh enzyme ciia dieu khien giam cua qua frinh sinh ting hgp F3 '5 'H Campanula hieu qua ban. anthocyanin. Tuy nhien, vi tinh frang bi kim bam ciia CHS CO the dan den viec tao ra cac cay trong chuyen Thuc hien cbuyen gen tren cay Lobelia erinus gen khong c6 flavonoid va ngudi ta nhan thdy rang mau hong vai mgt gen F3 '5 'H Lisianthus dudi su flavonoid dong vai fro quan trgng trong bao ve cay kiem soat cua mgt promoter CaMV35S tao ra boa co dual tac dgng cua tia UV, sy phong ve va trayen tin mau xanh. Cay Lobelia la mgt loai cay mo hinh biiu hieu cua cac loai thye vat noi chung (Winkel- ich cho cac ngbien cim ve bien doi mau sac vi n6 de Shirley, 2002); do do, su dieu khien giam cua gen dang thye bien cbuyen gen va hoa xuat bien frong CHS khong the la chien luge ly tudng dl phat trien mgt thai gian ngan chi sau 3-4 thang dong nuoi cay cac giong boa trang. That vay, ngudi ta da quan sat vai Agrobacterium mang cac vector chuyen gen cac cay co gen CHS bi ire che va nhan thay cac cay (Kanno et al, 2003). nay nhay cam ban doi vai stress. Sy dieu khiin giam cua cac gen khac trong chu frinh, nhu DPR bay F3H, Sy bieu hien cua F3 '5 'H Petunia frong mgt dong CO the la mgt sy thay the hgp ly ban de tao ra cac hoa cam chudng tich tu cac sac to c6 nguon goc tit giong cay hoa trang ma khong co cac tac dgng co cyanidin dan den su tao tbanh delphirudin d miic do hai. Tuy nhien, khi Zuker va dong tac gia (2002) rat thap va khong c6 anh buong dang ke nao den mau dieu khien giam F3H d cay hoa cam chudng, tac gia sac cua hoa (Bragliera et al, 2000). Dieu nay cho thay da phat hien ngoai su bien doi bam lugng rang F3 '5 H Petunia dugc dua vao khong the canh anthocyanin con co sy gia tang ham lugng franh mgt each bieu qua vai cac enzyme DFR va F3 'H methylbenzoate lam cho hoa tham ban, day co the ngi bao cua cay cam chudng. Tuy nhien, khi mgt gen xem la mgt ket qua tich cyc frong viec thuang mai cytochrome b5 Petunia va gen F3 '5 'H Petunia dugc boa cac cay hoa nay. Jorgensen va dong tac gia bieu bien frong cimg mgt dong cam chuang thi c6 sy (2002) kham pha rang sy ddng uc che cua F3'5'H cai thien dang ke ve mirc do san xuat delphinidin va hay DFR trong cay Petunia c6 the dan den sy bat CO sy thay doi mau sac cua hoa tir mau dom hong va dye cai, dieu nay co the do sy tich ty cua do sang mau dom boa ca va tia. dihydroflavonol trong lop vo hat. Cong ty Florigene va cong ty Suntory da nghien cim thanh cdng mgt loat nhiing cay cdm chuang tim Cac nghien ciiu tao ra hoa mau xanh chuyen gen bang each dua mgt gen F3 '5 'H vai mgt Hau het cac loai hoa mau xanh deu chiia dan gen DFR pemnia vao mgt cay cdm chuang mau xuat tham acylated delphinidin. Hoa hong, hoa cue frdng thilu DFR (Moi et al, 1998). Cac canh hoa cua va hoa cam chudng chiem ban 50% thi traang hoa cay cam chudng da qua xit ly chiia chii ylu cat canh tren toan the giai nhung chiing chi chiia cac delphinidin trong khi cac cay cdm chudng ty nhien dan xuat pelargonidin va cyanidin, cac dan xuat nay khong CO. Mau sac hoi xanh frong cac boa chuyin khong kit hgp vdi nhom acyl tham. Vi vay, chung gen khong bao gia co dugc a cay cdm chudng bdng fra thanh myc tieu cho cac no lyc nghien cim tong phuang phap chgn giong truyin thing. hgp cac dan xuat delphinidin vai hy vgng cuoi cung Flavonoid cua cac canh hoa Florigene la CO thl tao ra cac loai hoa mau xanh. Sy hap thu Moonshadow^'^ da dugc pban tich mgt each chi tilt cua anthocyanin hudng ve cac buac song dai (mau (Fukui et al, 2003). Cac canh boa cdm chudng tii xanh), khoang 10 nm (frong qua frinh hydroxyl boa nhien chu yeu cbiia pelargonidin hay cyanidin 3,5-0- vong B) va 4 nm (frong qua frinh acyl boa thom) diglucoside-6"-0-4, 6"'-0-l-cyclic-malyl diester. (Goto, Kondo, 1991). Hoa chuyen gen chiia delphinidin 3,5-0-diglucoside- Enzyme chinh trong qua trinb sinh ting hgp 6"-0-4,6"'-0-l-cyclic-malyl diester nhu sdc tl delphinidin la F3 '5 'H. Ngudi ta nhan thdy rdng cac chinh. Cac kit qua nay cho thdy cac enzyme tham 400
6. Tgp chi Cdng nghi Sinh hgc 7(4): 397-410, 2009 gia vao qua trinh sinb tong hgp anthocyanin d cay Cac nghien ciiu tao ra hoa co mau tir do den cam boa cam chudng du linh boat de co the bien doi cac DFR petiania khong co kha nang lam giam sac to CO nguon goc tir delphinidin. Cac canh hoa dibydrokaempferol do do hoa Petunia it khi chiia ciing chila apigenin 6-C-glucosyl-7-0-glucoside- anthocyanin dang pelargonidin va vi vay khong t^o 6"'-malyl ester, ngudi ta timg cho rang chat nay c6 ra mau cam din do gach. Cac cay Petunia chuyen hieu qua dong sac to manh. pH kliong bao ciia hoa gen mau do gach tich liiy anthocyanin dang Moonshadow da dugc thong ke nam trong khoang pelargonidin dugc tao ra do su bieu hien cua cac gen 5,5 bang each do pH cila nuac ep canh hoa. Vi vay, DFR tir cac loai khac nhau nhu cay bap, hoa dong mau sac xanh c6 the dugc giai thich la do su tich tu tien va hoa hong trong mgt dong Petunia dot bien cua nhiing anthocyanin dang delphinidin thong qua tich lay dibydrokaempferol (thilu F3'5'H, F3'H va su bieu bien cua gen F3'5'H, sy hien dien cua FLS). Viec xac dinh mgt dong tich IQy flavone, mgt dong sac to manh va pH khong bao dibydrokaempferol tuong ty trong so cac loai thuang canh boa tuong doi cao la 5,5. Cac dieu kien nay rat mai boa quan trgng la rat kho khan. Mizutani va thuan lgi cho viec xir ly de tao ra hoa mau xanh d dong tac gia (2003) da xir ly mgt dong Petunia do cac loai khac. tich liiy cac sac to c6 nguon goc tir cyanidin mgt Cac mile do dong sac to co the dugc bien doi each binh thudng de tao cac sac to c6 nguon goc tit thong qua cac ky thuat di trayen, vi cac gen FLS va pelargonidin (cam) bang sy dieu khien giam ciia gen FNS cQng da dugc tach chiet. Tuy nhien, vi F3 'H va su bieu hien cua mgt gen DFR hoa hong. flavonol va flavone c6 chung tien chat vol Nhieu loai boa quan trgng bao gom cay boa anh anthocyanin nen ham lugng cila dong sac to va thao, cay phi yen, cay hoa thuy tien, cay long ddm va anthocyanin thudng tuang quan nghich vai nhau. cay dia lan dugc dy doan la khong tich IQy Sy lie che antisense cua gen FLS a cay thuoc la dan pelargonidin do tinh chuyen biet co chat ciia DFRs den sy giam ham lugng ciia flavonol va sy gia tang ngi sinb cua chung. Cac each tuang ty vi the cung co ham lugng cua anthocyanin len den 3 lan (Holton et the dugc sir dung de tao ra hoa co rnau cam d cac al, 1993). Tuy nhien, sy dieu hoa giam cua FLS loai nay. petunia trong mgt cay Petunia tia dan den sy giam Ueyama va dong tac gia (2002) da sir dyng quy ham lugng cua flavonol nhung khong c6 sy thay trinh chuyen gen hai buac de tao hoa mau hong dam doi ham lugng cua anthocyanin. Nielsen va dong tir mgt cay Torenia hoa xanh. Ban dau, gen F3 '5 'H tac gia (2001) da tien hanh dieu khien giam FLS bi dieu khien giam de tao cay Torenia co hoa mau trong cay Lisianthus {Eustoma grandiflorum) bang hong. Qua tiinh chuyen gen tiep theo doi vai cay each Sll dyng antisense. Hoa tit cac cay chuyen gen torenia nay dugc thye hien vdi mgt promoter tich lily dihydroflavonol d phan tieu thu flavonol va CaMV35S khdi dgng gen F3'H torenia va marker dan den ket qua la mau sac cua hoa do ban. Sy hinh chgn lgc khac de tao hoa Torenia hong dam. thanh sac to do khac la ciing hien dien d giai doan choi sdm va nhuy hoa cGng c6 mau sac dam, ben Cac nghien cuu tao ra hoa mau vang c^lil^do, kieu hinh on dinh va dugc ke thita a cac Chalcone va aurone gop phan tao sac vang da the h?Cay thil hai. dugc quan sat d vai loai hoa. Chalcone thong dung Aida va dong tac gia (2000) nhan thay rang nhat la THC c6 mau vang nhung bi dong phan hoa hoa cua cay torenia chita mgt gen antisense DFR mgt each tu nhien thanh naringenin a frong ong xanh ban nhiing hoa chira gen antisense CHS vi sy nghiem va bi dong phan boa mgt each nhanh chong dieu khien giam khong hoan toan cua DFR dan frong CO the bai CHI (Jez et al, 2000). 6 cac hoa den sy tich tu flavone va tac dgng dong hinh thanh vang cua cay cam chuang, cay hoa mau dan va cay sac to vdi cac anthocyanin con lai se thay doi mau dita can, THC tich IQy nhu mgt 2'-glucoside sac boa theo chieu huang ve mau xanh. Day co the (isosalipurposide). Sy tich IQy ciia THC 2'-glucoside la each hUu ich de tao ra hoa mau xanh. Khi gen dugc cho la do tiang thai thieu hyt boat dgng cila FNSII trong torenia mau xanh bi dieu khien giam, CHI. Mgt nghien ciiu traoc day (Itoh et al, 2002) ham lugng ciia flavone giam va ham lugng ciia cho rdng ca gen CHI va DFR diu bi chen vao bdi tien chat flavanone tang. Tuy nhien, dieu bat nga mgt fransposon trong vai cay cdm chuang co mau la cac muc do cua anthocyanin giam va mau sac vang ldn vai mau dim va hinh quat trdng. Cay hoa hoa cuoi ciing la mau xanh nhat (Ueyama et al, anh thao mau vang nhat cung da dugc nhan thdy co 2002). tinh tiang thilu boat dgng cua CHI. Vi vay no thilu 401
7. Duong Tdn Nhyt et al. boat tinh CHI va sy hien dien ciia boat tinh UDP- flavonoid hay mgt gen dilu khien cua qua frinh sinh glucose (THC 2'-glucosyltransferase-C2'GT) can ting hgp ddn tai viec tao thanh cac hinh quat trang thiet cho sy tich luy cua isosalipurposide mau vang. trong mau nen cua hoa. Cac gen ma boa cho boat tinh C2'GT d cay cam Sy loai bo mgt fransposon nhu vay tii mgt gen chuang da dugc Okuhara pban lap vao nam 2004 chuyen biet thudng ddn din su tao thanh cac -viing co (Okuhara et al, 2004). Vi the cac thao tac ky thuat di trayen fren isosalipurposide a hoa bay gid da co the mau tren mgt nen frang. Liu va dong tac gia (2001) thye hien dugc. da tao ra cac hoa co dom mau thong qua cbuyen gen cay thuoc la bang each sir dyng mgt vector kep chila Aurone la cac flavonoid mau vang sang va vi the transposon Tagl cua cay Arabidopsis, fransposon cho den nay van chua c6 mgt gen dich dang quan nay dugc chen giua promoter CaMV35S va gen R tam nao khac dugc sir dyng cho cac thao tac ky thuat cua cay bap. Trong so cac cay Petunia chuyen gen di tmyen. Aurone dugc tim thay trong cac hoa mau dugc tao thanh, gen R, mgt gen dieu khien vang cua cac loai c6 quan he xa vol nhau bao gom (regulator) duong tinh frgi cua cac gen tham gia vao cay mom cho, cay thugc dugc, Limonium, cue qua trinb sinh tong hgp anthocyanin chi dugc phien zinnia, cay bim bim hoa tia. Con dudng sinh tong ma khi Tagl bi cat bo. Mgt nira trong so cac cay hgp aurone van con la mgt trong sd nhung dieu bi an trong chuyen gen co kieu binh hoa dang dom. Moi chua dugc giai quyet cua cac qua frinh sinh tong hgp dong CO mgt kieu binh khac nhau, vai cudng do mau flavonoid. Nakayama va dong tac gia (2000) da tinh sac cQng khac nhau. Viec xir ly cay hoa vdi che dugc aurone synthase, aureusidin synthase (AS) transposon co tao ra hoa mang gia tri thuang mai bay tir cac canh hoa cua cay hoa mom cho mau vang va khong van chua dugc xac dinh, tuy nhien xu hudng cDNA ma boa cho enzyme nay ciing da dugc tao cong nghiep bien nay van ua chugng cac dong on dong. dinh ban. 0 cac giong mau vang cua cay Asteraceae nhu NGHIEN CUU TAO RA HOA LAU TAN cay ciic van thg tay va cay thugc dugc, 6'- deoxychalcones la cac sac to chinh (Davies et al, Tuoi thg cua hoa sau thu hoach bi anh budng 1998). Qua frinh deoxyl boa d vi tri 6' cua THC dau tien bdi chat dinh duang, sy phat triln cua vi dugc xuc tac bai polyketide reductase (PKR) (ten ggi sinb yat, ethylene, cac dap ung khac va qua trinh lao chinh thiic la chalcone reductase) lam on dinb hoa. Loai hoa cat canh pho bien nhat tren thi trudng chalcone va tao mau sac vang. Davies va dong tac toan cau la hoa bong, hoa cam chuong va hoa ciic. gia (1998) da bilu bien mgt PKR cDNA tir cay Su tao thanh ethylene ngi sinh cua cac loai hoa nay Medicago sativa frong mgt dong Petunia frang va lam day nhanh qua frinh lao boa hoa d rieng cay cam thu dugc hoa mau vang nhat tich luy chalcone butein chuong. Sy ryng canh d boa hong, a mgt vai giong, 3-O-glucoside va butein 4-O-glucoside. Tuy nhien, CO the dugc thuc day bdi mgt loai ethylene ngoai mau sac chi co the thay dugc d cac ny hoa va khong sinh dac biet frong qua frinh van chuyin va tin tru du manh de dai dien cho mgt giong mau vang mai co boa, qua. Tat ca cac loai boa cat canh deu nhay cam gia fri thuang mai. Cac ket qua tuang ty cung da vai sy phat tiien cua vi sinh vat trong nuac cdm hoa dugc ghi nhan khi tien banh chuyen gen cay petunia a nhung nhiet do khac nhau ddn din tinb frang bao vdi mgt gen PKR cua cay cam thao. Joung va dong vay ciia mo mach lam ngan can sy di chuyin cua tac gia (2001) da bao cao rang sy bieu hien cua gen nuac trong than, va vi vay su heo tan xay ra lam PKR tit cay Pueraria montana trong cay thuoc la giam tuoi thg cua boa d trong binh. Cac vi sinh vat lam thay dii mau sdc hoa tit hong sang tiang do sy thudng bam vao hoa trong qua frinh san xudt nen co giam anthocyanin va su tong hgp flavonoid the xir ly bang each lam sach trong tdt ca cac cong liquiritigenin la. doan cua qua trinh xit ly sau tbu hoach, bao gIm ca Cac nghien cihi lam thay doi mau sdc hoa Idiac viec chuan bi nuac cam. Thilu chat dinh duang, chu yeu la dudng, cung co thl thiic ddy qua frinh lao hoa. Cac kilu hinh dim mau d hoa va la thudng dugc Sy thieu chat nhu vay co thl dugc cai thien bdng danh gia cao dii vai cac loai cay trang tri va da dugc each bl sung cac chat dinh duang vao dung dich nghien cilu d boa cua cay bim bim hoa tia tiong suot nuac cam. nhilu nam qua. Dang dim trong cac hoa nay thudng dugc gay ra do mgt transposon (lida et al, 1999). Sy Hoa cam chuang dugc xir ly sau thu hoach mgt chen cua mot sen nhay vao mgt gen sinh tong hgp each dac biet vai thiosulfate bac (STS). Viec xit ly 402
8. Tgp chi Cong nghe Sinh hgc 7(4): 397-410, 2009 nay co hieu qua khac nhau phy thugc vao thai gian, 1 gen dudi sy kilm soat promoter cua chinh no, nong do dung dich xil ly va loai hoa dugc xit ly. Ag* promoter CaMV35S la chu ylu hay promoter FBPl can trd mgt each huu bieu sy nhan biet ethylene cua (protein lien kit vai cay boa), cung da dugc tao ra. hoa (thong qua viec gan ket vai ethylene receptor Khoang nita trong sl cac cay hoa nay co qua trinh fren mang te bao), vi vay hoa khong co phan iing voi lao boa xay ra cham ban it nhdt la 6 ngay, thai gian ethylene ngi bao va ngoai bao. 0 cac cay boa cam cham hen cao nhdt la 16 ngay, tang gdp 3 lan thdi chuang dang canh chum vai nhieu hoa tren moi than, gian song trong binh. Thdi gian song trong binh thong thudng d cac giai doan khac nhau cua sy phat tham chi la dai ban so vdi hoa da dugc xil ly vai h6a trien, xuat hien bat lgi la mgt vai hoa se dugc tiep chat lie chi qua frinh sinh tong hgp ethylene hay sy xiic vdi It Ag"^ bon cac boa khac khi xit ly vol STS. phan ling dii vai ethylene (STS) (Bovy et al, 1999). STS la mgt hda chat dgc hai; khi ap luc doi boi phai Cac kit qua tirang tu dugc thu nhan bang each sir giam viec sir dung cac loai boa chat dgc hai trong dyng gen Etrl-I dugc dilu khiin bai promoter nong nghiep dang ngay cang gia tang, cung nhu cac CMB2 {CMB2 la mgt MADS box gen d cay cdm tien bg frong nhan thiic ve sy lao b6a cua hoa da tao chuang) (Baudinette et al, 2000). CO hgi cho cac nha di fruyen bgc nghien cim de lam Sy kim ham qua trinh bieu hien cua gen Etrl-l a cho qua frinh nang cao tuoi thg song ttong binh cila nhieu hoa da dugc chiing minh tao ra cung kieu hinh cay hoa cam chuang dugc keo dai ban ma khong can va CO mgt thuan lgi ve mat ly thuyet la sy can thiep CO boa chat. Tuy nhien, su thuang mai hoa san pham vao qua trinh nhan biet ethylene se lam giam dap nhu vay van chua thye hien dugc chu yeu la do chi img phong ve d thye vat. Ngudi ta van chua biet lieu phi ling dung frong ky thuat cQng nhu kien thiic ve rang day co phai la mgt van de doi vai cac cay bieu tac hai cua dgc chat van con ban che, vi vay hoa da bien Etrl-l chuyen biet a hoa hay khong nhung dieu dugc xu ly frong thdi gian dai nhung kho co the nay xem ra it co kha nang. thuyet phyc ngudi tieu thu mua san pham vdi gia thanh cao va su an toan chua dugc tham dinh. Mgt so cay trong frong chau co hien tugng rung canh hoa de dap ung doi vai ethylene va mgt chien Da CO mgt so each khac nhau nhung lai co lien luge da dugc thit nghiem nham cai thien hieu ilng quan vai nhau dugc sir dung de lam cho thdi gian nay. Sy bieu hien mo dac hieu cua gen bi dot bien cam trong binh cua hoa cam chuang dugc keo dai ehac chan se la mgt thuan lgi. Aida va dong tac gia ban ma khong can phai xit ly hoa chat. Dau tien lien (2000) da bao cao ve sy dieu khien giam cua AGO quan den sy dieu boa giam qua frinh tong hgp tiong hai giong Torenia fournieri. Tuoi thg boa trung ethylene a hoa cam chudng thong qua viec bat boat binh cua cay Torenia cbuyen gen vai ACO dugc gen dac trang cua hoa sau phien ma la gen ma boa dieu khien giam la 2,7 -7,1 ngay; frong khi tuoi thg ACC Oxidase (AGO) (Savin et al, 1995) hay ACC boa trang binh ciia cac cay hoang dai la 2,0 ngay. Synthase (ACS), la nhiing enzyme xiic tac hai buac Hon nQa, cac cay Torenia chuyen gen vai doi song cuoi cung trong qua trinb sinb tong hgp ethylene. hoa dugc keo dai tao nhieu hoa cung mgt luc tren Dieu nay lam hoa mat do nhay cam doi vai ethylene moi than ban loai cay hoang dai. Dac tinh thdi gian ngoai bao mac dil thdi gian song trong binh co the so song cua hoa keo dai sang the he con chau lien be sanh dugc vdi thai.gian khi than dugc xit ly vai STS den su ton tai cua gen. sau tbu hoach. Tuy cac ham lugng ethylene ngoai bao chua dugc xem la mgt hau qua frong chuoi van Khi gen EtrI-1 dugc chuyen vao cay Petunia chuyen hoa cam chuang nhung ngudi ta van cho dudi sy kiemisoat cua mgt promoter CaMV35S dugc rang san pham kem hap dan hon cac than cay da tang cudng, cac hoa Petunia chuyen gen co thdi gian dugc xu ly b6a chat. Sy dien giai ve con dudng dan song keo dai (dai ban tir 2 den 4 lan) va tii hoan su trayen ethylene d Arabidopsis (Fluhr, 1998) giiip rung canh so vai cac doi chiing khong chuyin gen. phan lap mgt gen ma hda ethylene receptor tit Chimg cung khong nhay cam doi vol ethylene ngoai Arabidopsis (Efrl), sau do qua frinh san xuat hoa bao ma tong hgp nhieu ethylene ban (Wilkinson et cam chuang vol thdi gian cam keo dai khong can al, 1997). Gen Etrl-I dual sy kiem soat cua mgt boa chat va khong nhay cam doi vai ca ethylene ngi protein lien ket cua cay hoa (FBPl) bay mgt bao va ngoai bao da dugc thye hien thong qua viec promoter apetala (AP3) da dugc dua vao cay dua vao mgt gen ma boa ethylene receptor petunia. Han 60 cay Petunia chuyin gen da thu dugc Arabidopsis da bi dot bien (Etil-1). ling vai mli loai tren. Ldn lugt khoang 70% va 30% cay CO thdi gian sing cua hoa gdp hai ldn thdi gian Cac cay hoa cam chudng cbuyen gen chira Etrl- song cua hoa Petunia khong chuyin gen. Mgt sl cay 403
9. Duong Tan Nhyt et al. CO gen Etrl-l dugc dieu hoa bdi FBPl promoter co bdi CaMV35S promoter tao ra cac bien doi hinh thai hoa nd deu dan trong suot 14 ngay trong khi hoa khac nhau nhu chilu cao cay, kich thuoc la va hoa khong chuyen gen chi nd frong 3 ngay (Cobb et al, giam, sy phan nhanh tang (Winefield et al, 1999). 2002). Cac gen rolA, B va C dugc chuyen vao cay Rosa hybrida cv. Moneyway lam cac dac diem ra re ciia Khi cay Petunia dugc thye hien cbuyen gen vol cay dugc cai thien (Van der Saim et al, 1997). mgt gen ERS bi dot bien (mgt ethylene receptor gen) cua cay Brassica oleracea, hoa cua cac cay chuyen Gen CENTRORADIALIS (CEN) cua cay hoa gen giu dugc do cang va sac to lau hon hoa cua cac mom cho ma boa cho mgt protein tuong dong lien cay khong cbuyen gen va khong nhay cam doi vai ket vai phosphatidyl ethanolamine va can cho sy ethylene ngoai bao. Cay chuyen gen tao ra hoa to phat trien vo ban cua re. Mat khac, cay thuoc la co ban nhung c6 ti le chet cao ban; dieu nay cho thay cac mo pban sinh re giai ban do sy cbuyen b6a thanh cac cay Petunia khong nhay cam doi vai ethylene thi cac mo pban sinb hoa. Cac cay thuoc la bieu bien nhay cam ban doi vai dich benh (Shaw et al, 2002). vugt nguang CEN co mgt pha sinh trudng keo dai vai nhieu la hon va kieu phat trien cao bon, fri hoan sy phat sinh hoa frong suot ban 10 thang (Amaya et NGHIEN Ciru BIEN DOI HINH THAI HOA al, 1999). Cac boa bong hien dai nhu cac giong cay thao moc va fra lai thudng xac dinh nhung cac thay Nhieu gen tiem nang co lgi lien quan den cac con doi dot bien khong xac dinb thudng phat sinh d cac dudng phat sinb hinh thai cua boa va cay da dugc tao cay boa bong leo. Iwata (2003) da xac dinh mgt gen ddng. Cac nhan to phien ma dieu khien qua trinh sinh nhay dugc chen vao mgt the tuong dong frong bg tong hgp va phat trien d thye vat hay cac gen dieu gen cua cac hoa hong xac dinh. Sy loai bo khien bao gom cac hormone thye vat la cac gen fransposon dan den sy chuyen doi cac hoa hong xac thudng dugc quan tam. Tuy nhien, chi mgt vai gen dinb thanh cac loai hoa hong leo khong xac dinh. frong so nay da dugc iing dung thye sy vao cac cay Cac ket qua nay cho thay rang sy dieu boa tang hay hoa. Tham chi frong vai traang hgp nay, cac promoter giam cua CEN va cac the khong tuang dong cua no thiet yeu dugc sir dyng, thudng tao ra cac loai cay CO the dugc ilng dung de thay doi chieu cao va kien khong dugc ua thich. Sy dieu khien phitc tap hon tnic ciia cay. ttong su bieu hien cua cac gen nay co the tao ra cac cay hoa vai hmh thai la co tiem nang thuong mai. Cac boa chat nhu uniconazole dugc sir dyng Kiem soat sy pban nhanh dugc xem la mgt each rgng rai de tao ra cac cay lim. Nhieu gen lien quan tot nhat de tao cay c6 dac tinh la hap dan. Sy bieu den qua trinh sinh tong hgp va nhan biet gibberellin hien qua nguang cua nhan to phien ma co dang ngon da dugc phan lap (Olszewski et al, 2002). Trong so tay kem a cay Petunia, nhan to cam iing choi ben chung, mgt alen dot bien co chung tinh trang frgi cua GAI la gai-1 lam giam dang ke sy dap ung doi vai (LIF) trong cay Petunia duai su kiem soat cua mgt gibberellic acid trong suit qua trinh phat friln sinh CaMV35S promoter dan den sy gia tang dang ke so trudng. Gen nay c6 the dugc sit dung de tao ra cac lugng nhanh. Cac nhanh thii cap it khi binh tbanh cay lim nhu Suntory Ltd. da thanh cong frong viec trong cac cay Petunia hoang dai lai xuat bien trong tao ra cac cay lim bang each dua cac trinh ty gai-1 cac cay chuyen gen nay. Ham lugng cac cytokinin ty vao cay Petunia. do thap ban trong khi cac the nucleotide va nucleoside cua chiing cao ban a than va la. Sy dilu khien cytokinin thye su quan frgng nlu muc dich la NGHIEN CUU BIEN DOI MUI HUONG CUA gia tang so lugng hoa. Cay thuoc la cbuyen gen dugc HOA thye hien vol mgt gen Agrobacterium ipt (isopentenyl fransferase) duai sy kiem soat ciia mgt Mui buong cua hoa dong vai tro quan frgng frong promoter lien quan den sy lao boa a la Arabidopsis viec hap dan cac loai con trimg thy phan. N6 cung tao ra nhieu hoa ban do su lao boa la bi fri hoan quan frgng den sy lya chgn hoa cua khach hang do no (Gan, Amasino, 1995). Sy tri hoan qua trinh lao boa lien quan den sa thich cua khach bang. Mui huong trang hoa d cay Petunia da dugc bao cao vai mgt cau ciia boa dugc tao thanh tit nhieu hgp chdt khac nhau. tnic gen xac dinh (Chang et al, 2003). Hon 700 hgp chat da dugc xac dinb d 60 hg thye vat Gen rolC tir Agrobacterium rhizogenes ma boa (Knudson et al, 1993). Chung la cac ddn xudt acid cho cytokinin -(i-glucosidase. Cay Petunia cv beo nhu benzenoid, phenylpropanoid va terpenoid Mitchell chuyen gen bieu bien rolC dugc dieu khien (monoterpene va sesquiterpene). Cac cdu tnic cua 404
10. Tgp chi Cdng nghi Sinh hgc 7(4): 397-410, 2009 hang fram hgp chdt thom nay da dugc xac dinb. Mac khong can su bien dien cila cac cau tnic nay. dil so lugng cac gen dugc tao dong lien quan den qua Thl lai petimia Wl 15 dugc biln dii vai Clarkia trinh sinh tong hgp cac hgp chat tao mui huong gia LIS cDNA dudi sy kilm soat ciia promoter tang mgt each lien tyc nhung nhung kien thite sinh CaMV35S. Hoat tinh enzyme thich hgp da dugc tim hgc phan til va sinh hoa cua qua tiinh sinh tong hgp thay trong la va canh hoa cua cay chuyen gen. Tuy cac hgp chat nay van rat ban che (Dudareva et al., nhien, chi co mgt sl lugng rat nho S-linalool dugc 2003). Cac bao cao ve su bien doi cua mui huong d phat hien frong hoa. Dilu nay cho thay hau bet S- hoa bang each sit dyng ky thuat di tiuyen tham chi con linalool dugc tong hgp bi bien doi thanh S-linalyl-p- hiem ban. Ndi chung, cac hgp chat mui huong d hoa D-glucopyroanoside khong bay hoi, c6 the la do boat dugc tong hgp de novo frong cac canh hoa. Cac dgng cua glucosylfransferase ngi sinb a cay petunia. enzyne lien quan den qua frinh sinh tong hgp mui Lugng S-linalool va glycoside cua no dudng nhu huong hoa bieu hien mgt each manh me frong cac te phu thugc vao kha nang ciia co chat GDP nhieu ban bao bieu bi canh hoa va qua trinh bieu bien ciia chung mile do bieu bien cua gen LIS (Lucker et al, 2001). dugc dieu hoa a mile do phien ma phu thugc vao giai Gen tuang ty duai sy kiem soat cua CaMV35S doan phat trien canh hoa. promoter dugc dua vao cay cam chuang {Carnation Cac hoa Petunia truang thanh chu yeu giai cv. Eilat) tao cac dan xuat benzoic acid va phong benzoids. Su toa huang nay xuat hien nhip sesquiterpene nhung khong tao monoterpene mgt nhang 1 lan/ngay, tien tdi cue dai vao liic toi. Cac each binh thudng. Cac cay cbuyen gen tao thanh san chat bay bai nay khong dugc bao ton trong cac thdi xuat linalool va cac dan xuat cua no, cis- va trans- ky lan toa thap nhung phan nao dugc tong hgp de linalool oxide. Tuy nhien, sy lan toa cua linalool novo. Phan tich DNA-microarray Cho thay rang cac khong xay ra d mgt miic do ma co nhiing bien doi gen cua chu trinh dan den sy tao thanh benzoid dugc mui huong d hoa dugc ghi nhan bdi thinh giac cua dieu hoa tang trong ngay traoc khi gia tang sy lan con ngudi (La-vy et al, 2002). Khi gen tuong tu toa cua chiing (Verdonk et al, 2003). Su tong hgp dugc bieu hien frong frai ca chua, lugng S-linalool cac chat bay hoi d hoa cam chuong dugc dieu boa tao ra dii de thinh giac con ngudi co the phat hien mgt each dien bien va ngudi ta nhan thay rang qua dugc (Lewinsohn et al, 2001). Cac bao cao nay cho frinh tong hgp dugc lien ket vdi mang te bao va su thay rang sy bien doi mui huong co the thye hien pban chia vao cj^osol dien ra mgt each phu hgp vai dugc bang each chuyen mgt gen ma boa cho mgt cac he so phan chia (Schade et al, 2001). Cac ldp enzyme sinh tong hgp va mgt chu frinh boat dgng cutin a canh hoa cay hoa mom cb6 bau nhu khong cua enzyme do, de cung cap mgt bam lugng -Vila du tao ra bat cii kha nang khang khuech tan nao doi vdi cac chat tham gia can thiet (trong tradng hgp niy la cac chat bay hoi va vi vay cho phep lan toa mgt each geranyl pyrophosphate). nhanh chong cac hgp chat nay (Goodwin et al, 2003). Cac ket qua nay cho thay sy bieu hien cua gen Hai gen ma hoa cho acetyl CoA (benzylalcohol sinh tong hgp mui huong 6 cay chuyen gen co the acetyltransferase) va benzyl CoA (benzylalcohol dan den su bien doi buong tham cua hoa. benzoyl transferase), la hai enzyme chiu trach nhiem tong hgp benzylacetate va benzylbenzoate, lien quan den qua trinh sinh tong hgp mui huong da dugc tao Gen cau tnic dau tien dugc pban lap ma boa cho dong tit C breweri va sau d6 2 gen nay da dugc xac mgt enzyme sinh tong hgp mui buong hoa la S- linalool synthase (LIS) tir Clarkia breweri, mgt cay dinh (Dudareva et al, 2003). Gen ma hoa cho mgt ty nhien d California toa mui thom manh co S- acetyl CoA (alcohol acetyltransferase) tir cay dau tay Linalool la mgt thanh phan chinh cua no. S-Linalool dong vai tio chu yeu tiong viec san sinh mui vi frong dugc sinh tong hgp tir geranyl pyrophosphate, mgt suot qua frinh chin ciia qua (.'Uiaroni et al, 2000) va chat trang gian cua nhieu terpenoids khac nhau nha mgt acetyl CoA (geraniol acetyltransferase) tii cay LIS. Gen LIS dugc bieu bien cao trong cac te bao boa bong cung nhan nhdm alcohol nhu citronellol va bieu bi ciia cac canh hoa va frong cac te bao cua 1-octanol lam co chat (Shalit et al, 2003) da dugc •vung trayen d dau nhuy va voi nhyy; lugng protein tao dong. tao ra dugc sit dung vao dieu boa phien ma. Ngudi ta Cac hoa cila cay hoa Torenia toa ra mgt thanh cung nhan thay rang cac tuyen thom cbuyen biet va cac ca quan lien quan khong can thiet doi vai sy tao phan tao miii hoa phenylpropanoid chu ylu, tbanb mui buong a boa (Dudareva et al, 2003), dieu methylbenzoate. SAdenosyl-L-metbionine:benzoic nay cho thay mui huang d boa co the bi bien doi ma acid carboxyl methyl transferase (BAMT) xiic tac buac culi cilng frong qua frinh sinh ting hgp methyl 405
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