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Báo cáo " Phân lập gen 4CL1 từ cây thông đuôi ngựa (Pinus massoniana Lamb.) trồng tại Việt Nam "

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Phân lập gen 4CL1 từ cây thông đuôi ngựa (Pinus massoniana Lamb.) trồng tại Việt Nam

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Tgp chi Cdng nghe Sinh hgc 7(4): 479-483, 2009 P H A N L A P G E N 4 C L 1 TlT C A Y T H O N G D U O I NGU^A {PINUS MASSONIANA LAMB.) T R O N G TAI VIET N A M Ha Van Huan', H6 Van Giang*, Nguyen Nhu- Ngoc\ Bui Van Thang\ Nong Van Hai^ Chu Hoang Ha^ 'Truang Dgi hgc Ldm nghiip ^Vien Cdng nghi sinh hgc TOM TAT Nham tao nguon vat lieu phuc vu cho tao giong cay lam nghiep bien doi gen co chat lugng go tot, chiing toi da tien hanh phan lap gen 4CL1 tir doi tugng Thong duoi ngua (Pinus massoniana Lamb.) trong tai Viet Nam. Tach chiet mRNA long so tir tang xylem thii cap cua cay Thong duoi ngira su dung lam khuon de tong hgp cDNA sgi don. Gen 4CL1 dugc nhan len bang ky thuat PCR tu cDNA sgi don nho cap moi dugc thiet ke dua tren trinh tu nucleotide cua gen 4CL1 a cay Thong lira (Pinus taeda) da dugc cong bo tren Ngan hang gen (ma so PTU12012). San pham PCR nhan gen 4CL1 tir cDNA sgi don dugc tach dong nho vector tach dong pCR2.1 ciia hang Invitrogen. Cac dong te bao chita plasmid tai to hgp mang gen quan tam dugc sang lgc bang cac ky thuat nhu: nuoi cay tren moi truong chgn lgc, tach chiet plasmid, cat kiem tra plasmid bang enzyme han che va giai trinh tu nucleotide. Phan tich trinh tu nucleotide ciia gen 4CL1 cho thay toan bg viing khung dgc (ORF) mang ma di tmyen gom 1614 nucleotide. So sanh trinh tu nucleotide cua gen 4CL1 o cay Thong duoi ngira va cay Thong lira co ty le tuong dong 98%, trinh tu amino acid tuong dong 99%. Trinh tu nucleotide ciia gen 4CL1 ma chimg toi phan lap dugc tir cay Thong duoi ngua (Pinus massoniana Lamb.) da dugc dang ky tren Ngan hang gen (ma so FJ810495). Tie khoa: Cdi thien chdt lupng go, gen 4CL, ligin, Thdng duoi ngua, 4-coumarate:coenzyme A ligase GIOI THIEU Gen 4CL1 da dugc nghien ciiu phan lap tir mgt so doi tugng nhu: Bach duong. Bach dan. Thong lua, Hien nay, cac nghien ciiu ve cai thien chat lugng Arabidopsis,... (Zenn et al, 2006). Trong ngi dung go cay iam nghiep thuang dugc tien hanh theo hai nghien ciiu nay, chiing toi tien hanh phan lap gen huong chinh: tang ham lugng lignin cho muc tieu lay 4CL1 tu cay Thong duoi ngua {Pinus massoniana go lam do moc, thii cong my nghe, xay dung... va Lamb.) trong tai Viet Nam, nham cung cap nguon iam giam ham lugng lignin cho muc tieu lay go lam vat lieu cho cac nghien ciiu tao giong cay trong bien nguyen lieu cho cong nghiep bgt giay va sgi doi gen theo huong cai thien chat lugng go cay rimg. (Cukovic et al, 2001). Nhu vay, viec tao giong cay trong iam nghiep bien doi gen theo huong lam tang hay giam lignin la phu thugc vao muc tieu sir dung VAT LIEU VA PHUONG PHAP NGHIEN Cim go nguyen lieu. Ca hai huong nghien ciiu deu tap trung tac dgng vao cac gen ma hoa cho cac enzyme chia khoa co lien quan den sinh tong hgp lignin. Vat lieu Thong duoi ngua {Pinus massoniana Lamb.) Gen 4CL1 ma hoa cho 4-coumarate:coenzyme A ligase la enzyme chia khoa co vai tro dac biet quan dugc trong o rimg thuc nghiem (niii Luot) cua trgng cho sinh tong hgp cac hgp chat thii sinh, dac truang Dai hgc Lam nghiep. Cay lay mau c6 do tuoi biet la lignin 6 thuc vat (Kumar et al, 2003). Lignin tir 5 - 10 tuoi, sinh truang tot, khong bi sau benh. la hgp chat polymer thom, tham gia vao cau triic thanh te bao, dac biet la cac te bao cua mach xylem Hoa chat (Lu et al, 2003). Trong tl bao thuc vat, lignin c6 vai Bg hoa chat chuan de tach RNA tong so tir thuc tro gan kit cac sgi polysaccharide thong qua cac kieu vat "PureLink™ Plant RNA Reagent" (Invitrogen - lien kit hoa hgc khac nhau, giu cho thanh tl bao My); Kit tach chiet mRNA "mRNA magnetic bead •vimg chSc, nang cao kha nang chiu luc co hgc va kit micro" (Invitrogen - My); Kit tong hgp bao ve cac sgi cellulose khong bi phan huy boi cac cDNA "superscript III 1st strand" (Invitrogen - My); nhan t6 hoa - sinh hgc (Lu et al, 2004). Kit tach dong gen "TA cloning kit" (Invitrogen - 479
Ha Van Huan et al. My); Kit xac djnh trinh tu- nucleotide "BigDye*^ plasmid cho cac nghien ciiu tilp theo. Cac dong Terminator v3.1 Cycle Sequencing"; Chiing vi khuan plasmid dugc cat kilm tra bang cap enzyme han che E.coli TOP 10, moi truang nuoi cSy vi khuan LB, cac BamHl/Sacl va dien di tren gel agarose 1%. Trinh tu loai enzyme han che va mgt so hoa chat thong nucleotide cua gen 4CL1 dugc xac dinh bang thuang khac do cac hang: Invitrogen (My), phuang phap xac dinh trinh tu tu dgng tren may Reseachorganics (My), Fermentas (Diic) va Wako ABI PRISM"^ 3100 - Avant Genetic Analyzer (ABI, (Nhat) cung cap. My) tai Phong thi nghiem trgng dilm Cong nghe gen - Vien Cong nghe sinh hgc. Phu'OTig phap nghien cihi Tdch mRNA tdng so vd tong hap cDNA KET QUA VA T H A O L U A N Phuang phap tach mRNA tong so va tong hgp cDNA dugc thuc hien theo huang dan cua cac bg Kit: Tach chilt RNA PureLink'T'^ Plant RNA Reagent (Invitrogen - My), mRNA magnetic bead kit micro (Invitrogen - My) va De thu nhan dugc cac phan tir mRNA dam bao Superscript III 1st strand (Invitrogen - My). tieu chuan ky thuat lam khuon cho tong hgp cDNA, truoc het can tach chiet RNA tong so tir mau nghien Nhan gen 4CL1 tie cDNA ciiu. Ket qua tach RNA tong so dugc kiem tra bang Cap moi (primers) nhan gen 4CL1 tir cay Thong dien di tren gel agarose \%. Ket qua thu dugc duoi ngua dugc thiet ke dua tren trinh tu nucleotide (khong dua anh) cho thay: sit dung bg hoa chat cua gen 4CL1 o cay Thong lira {Pinus taeda) da chuan "PureLirLk^"'^ Plant RNA Reagent" cua hang dugc cong bo tren Ngan hang gen (ma so Invitrogen de tach chiet RNA tong so hi cay Thong PTU12012). Cap moi co trinh tu nucleotide nhu sau: duoi ngua dat hieu qua rat cao. RNA tong so thu Moi xuoi: 4CLlPinF: 5'- gcggatccgccatggccaac dugc dam bao chat lugng de tien hanh cac nghien ggaatcaa -3', co thiet ke trinh tu cat cua enzyme han ciiu tiep theo. Vi mRJSfA tong so chi chiem mgt ham chi BamlTl (GGATCC) a dau 5'. M6i ngugc: lugng rat nho (khoang tir 1 - 3%) trong RNA tong 4CLlPinR: 5'- gcgagctcttttcattttgctgccagtct -3', co so, ty le nay phu thugc vao loai mo, tuoi mo, thai thiet ke trinh tu cat cua enzyme han che Sad gian lay mau de tach RNA. Mat khac, trong dung (GAGCTC)adau5'. dich RNA tong so con chiia nhieu cac hgp chat thir sinh C the gay anh huang den ket qua tong hgp O D I nhan gen 4CL1 bang ky thuat PCR, cDNA cDNA. Do do, neu sii dung true tiep RNA tong so de sgi don dugc dimg iam khuon. Phan img nhan gen tong hgp cDNA thuang gap nhieu kho khan. De dugc thuc hien trong tong the tich 50 |il vai cac nang cao hieu qua tong hgp cDNA, can tien hanh thanh phan: DEPC treated water: 38 ^il; lOX PCR tinh sach mRNA. mRNA dugc tinh sach tir dich buffer minus Mg^"": 5 |al; 50 mM MgClj : 1,5 ^.1; 10 RNA tong so bang Kit "mRNA magnetic bead kit mM dNTP rnix: 1 |.il; 10 pM m6i xuoi 4CLlPinF: 1 micro" cua hang Invitrogen. Tinh sach mRNA dam ^1; 10 pM mSi ngugc 4CLlPinR: 1 ^1; dich cDNA: 1 bao cac tieu chuan ky thuat dugc dimg lam khuon de |j.l; Taq DNA polymerase ( 5 U/|a,l): 0,5 ^1. Phan tog tdng hgp cDNA. dugc thuc hien theo chuang trinh: 94°C trong 3 phiit; (94°C trong 45 giay, 56°C trong 45 giay, 72°C trong Tong hop cDNA va nhan gen 4CL1 1 phiit, lap lai 40 chu ky); u a 72°C trong 8 phut; san pham PCR dugc bao quan a 4°C. Sir dung cDNA sgi don dugc tong hgp tit Sdng lgc vd xdc dinh trinh tu nucleotide eda gen mRNA bang Kit "Superscript III 1st strand" dung 4CL1 lam khuon dl nhan gen 4CL1 bang ky thuat PCR San pham PCR dugc gan vao vector tach dong nho cap m6i 4CLlPinF va 4CLlPinR. Kit qua nhan pCR2.1 TA nho enzyme noi T4 DNA ligdse, sau do gen dugc kiem tra bang dien di treli gel agarose 1% . dugc bien nap vao te bao kha bien E. coli chung Tren dien di d6 (Hinh 1) chi c6 mgt bang duy nhat, TOP 10. Te bao vi khuan sau khi bien nap, dugc cay C kich thuoc khoang 1,6 kb tuang duong vai kich O trai va nuoi tren moi truong LB dac co b6 sung X-gal thuoc cua gen 4CL1 theo ly thuyet. Nhu vay, buac va khang sinh ampicillin (100 ng/ml) de tach thanh dau C the ket luan da nhan dugc gen 4CL1, cap m6i O timg dong rieng biet. Cac dong vi khuan dugc nhan dimg dl nhan gen 4CL1 tii cDNA sgi dan la rat dac len lugng lan trong moi truang LB long de tach chiet hieu. 480
Tgp chi Cdng nghi Sinh hgc 7(4): 479-483, 2009 M 1 2 3 dong con lai. Dilu do co nghia la cac dong plasmid nay da dugc chen them doan DNA ngoai lai (co the la gen quan tam). Cac dong P2, P3 va P5 co kich thuac tuang duong voi kich thuac ciia vector pCR2.1 nen loai bo. De C C so ket luan cac dong PI, P4, P6 va P7 O O 1636 b p . San pham mang gen quan tam, cac dong plasmid nay dugc cat PCR bang cap enzyme han che BamHl/Sacl. Ket qua dien di san pham cat (khong dua anh) cho thay cac dong plasmid PI, P4, P6 va P7 deu co bang tuang duang vai kich thuac cua san pham nhan gen 4CL1 va tuong duang vai kich thuoc cua gen 4CL1 (1,6 kb). Nhu vay, buoc dau co the ket luan cac dong plasmid Hinh 1. Ket qua nhan gen 4CL1 tif cDNA. U. Thang DNA tai to hgp PI, P4, P6 va P7 mang gen quan tam 4CL1. chuan 1 kb; 1. Doi chirng am; 2, 3. San pham nhan gen 4CL1 tif cDNA. Xac dinh trinh tu- nucleotide ciia gen 4CL1 De khang dinh chae chan cac dong plasmid phan Sang loc gen 4CL1 lap dugc mang gen muc tieu 4CL1, chiing toi tien san pham nhan gen 4CL1 tit cDNA dugc ghep hanh xac dinh trinh tu nucleotide cua doan DNA noi vao vector tach dong pCR2.1 TA de tao vector trong vector tach dong (pCR2.1-4CLl). Trinh tu tai to hgp pCR2.1-4CLl. San pham ghep noi dugc nucleotide cila doan chen dugc phan tich va so sanh bien nap vao te bao kha bien E.coli chiing TOPIO. vai trinh tu nucleotide cua gen 4CL1 o cay Thong Sau khi bien nap, vi khuan dugc cay trai tren moi lira {Pinus taeda) da dugc cong bo tren ngan hang truang LB dac bo sung khang sinh ampicillin va X- gen (ma so PTU12012). Ket qua dugc the hien tren gal. Cac dong te bao vi khuan sinh truang dugc tren hinh 2. moi truang co khang sinh chiing to mang plasmid chiia gen khang khang sinh. Chiing toi tien hanh Phan tich trinh tu nucleotide cua gen 4CL1 a cay chgn 7 khuan lac trang (ki hieu tir PI - P7), 1 khuan Thong duoi ngua {Pinus massoniana Lamb.) cho lac xanh (ki hieu: Px) cay vao 3 ml moi truang LB thay toan bg -VTing khung dgc (ORF) mang ma di bo sung ampicillin (100 ng/ml), nuoi a 37°C qua truyen gom 1614 nucleotide. Ket qua so sanh trinh tu dem. DNA plasmid dugc tach ra khoi cac dong tl nucleotide ciia gen 4CL1 a cay Thong duoi ngua va bao vi khuan E. coli va dugc kiem tra bang dien di cay Thong lira c6 ty le tuong dong 98%, trinh tu tren gel agarose P/o. amino acid tuong dong 99%. Do gen 4CL1 ian dau tien dugc phan lap tit cay Thong duoi ngua, nen Ket qua thu dugc (khong dua anh) cac dong chiing toi tien hanh dang ky trinh tu nucleotide ciia plasmid PI, P4, P6 va P7 co kich thuac lan hon cac gen nay tren Ngan hang gen (ma so FJ810495). 4CL1_P.TAEDA ATGGCCAACGGAATCAAGAAGGTCGAGCATCTGTACAGATCGAAGCTTCCCGATATCGAG 4CL1_P.MASSONIANA ATGGCCAACGGAATCAAGAAGGTCGAGCATCTGTACAGATCGAAGCTTCCCGACATCGAG 4CL1_P.TAEDA ATCTCCGACCATCTGCCTCTTCATTCGTATTGCTTTGAGAGAGTAGCGGAATTCGCAGAC 4CL1_P.MASSONIANA ATCTCCGACCATCTGCCTCTTCATTCGTATTGCTTTGAGAGAGTAGCGGAATTCGCAGAC 4CL1_P.TAEDA AGACCCTGTCTGATCGATGGGGCGACAGACAGAACTTATTGCTTTTCAGAGGTGGAACTA 4CL1_P.MASSONIANA AGACCCTGTCTGATCGATGGGGCGACAGACAGAACTTATAGCTTTTCAGAGGTGGAACTG 4C1,1_P. TAEDA ATTTCTCGCAAGGTCGCTGCCGGTCTGGCGAAGCTCGGGTTGCAGCAGGGGCAGGTTGTC 4CL1_P.MASSONIANA ATTTCTCGCAAGGTCGCTGCCGGTCTGGCGAAGCTCGGGTTGCAGCAGGGGCAGGTTGTC 4CL1__P. TAEDA ATGCTTCTCCTTCCGAATTGCATCGAATTTGCGTTTGTGTTCATGGGGGCCTCTGTCCGG 4CL1_P.MASSONIANA ATGCTTCTCCTTCCGAATTGCATCGAATTTGCGTTTGTGTTCATGGGGGCCTCTGTCCGG 4CL1_P.TAEDA GGCGCCATTGTGACCACGGCCAATCCTTTCTACAAGCCGGGCGAGATCGCCAAACAGGCC 4CL1 P.MASSONIANA GGCGCCATTGTGACCACGGCCAATCCTTTCTACAAGCCGGGCGAGATCGCCAAACAGGCC 481
Ha Van Huan et al. 4CL1_ P.TAEDA AAGGCCGCAGGCGCGCGCATCATAGTTACCCTGGCAGCTTATGTTGAGAAACTGGCCGAT 4CL1_ P.MASSONIANA AAGGCGGCGGGCGCGCGCATCATAGTTACGCTGGCGGCTTATGTTGAGAAACTGGCCGAT 4CL1^ P.TAEDA CTGCAGAGCCACGATGTGCTCGTCATCACAATCGATGATGCTCCCAAGGAAGGTTGCCAA 4CL1_ P.MASSONIANA CTGCAGAGCCACGATGTGCTCGTCATCACAATCGATGATGCTCCCAAGGAAGGTTGCCAA 4CL1_ P. TAEDA CATATTTCCGTTCTGACCGAAGCCGACGAAACCCAATGCCCGGCCGTGAAAATCCACCCG 4CL1_ P.MASSONIANA CATATTTCCGTTCTGACCGAAGCCGACGAAACCCAATGCCCGGCCGTGAAAATCCACCCG 4CL1_ P..TAEDA GACGATGTCGTGGCGTTGCCCTATTCTTCCGGAACCACGGGGCTCCCCAAGGGCGTGATG 4CL1_ P..MASSONIANA GACGATGTCGTGGCGTTGCCCTATTCTTCCGGAACCACGGGGCTCCCCAAGGGCGTGATG 4CL1_ P..TAEDA TTAACGCACAAAGGCCTGGTGTCCAGCGTTGCCCAGCAGGTCGATGGTGAAAATCCCAAT 4CL1_ P..MASSONIANA TTAACGCACAAGGGCCTGGTGTCCAGCGTTGCCCAGCAGGTCGATGGTGAAAATCCCAAT 4CL1_ P, .TAEDA CTGTATTTCCATTCCGATGACGTGATACTCTGTGTCTTGCCTCTTTTCCACATCTATTCT .MASSONIANA 4CL1_ P, CTGTATTTCCATTCCGAGGACGTGATACTCTGDGTCTTGCCTCTTTTCCACATCTATTCT 4CL1_ P,.TAEDA CTCAATTCGGTTCTCCTCTGCGCGCTCAGAGCCGGGGCTGCGACCCTGATTATGCAGAAA 4CL1_ P .MASSONIANA CTCAATTCGGTTCTCCTCTGCGCGCTCAGAGCCGGGGCTGCGACCCTGATTATGCAGAAA 4CL1_^P .TAEDA TTCAACCTCACGACCTGTCTGGAGCTGATTCAGAAATACAAGGTTACCGTTGCCCCAATT 4CL1 P .MASSONIANA TTCAACCTCACGACCTGTCTGGAGCTGATTCAGAAATACAAGGTTACCGTTGCCCCAATT 4CL1^ P .TAEDA GTGCCTCCAATTGTCCTGGACATCACAAAGAGCCCCATCGTTTCCCAGTACGATGTCTCG 4CL1_ P .MASSONIANA GTGCCTCCAATTGTCCTGGACATCACAAAGAGCCCCATCGTTTCCCAGTACGATGTCTCG 4CL1 P .TAEDA TCCGTCCGGATAATCATGTCCGGCGCTGCGCCTCTCGGGAAGGAACTCGAAGATGCCCTC 4CL1_ P .MASSONIANA TCCGTCCGGATAATCATGTCCGGCGCTGCGCCTCTCGGGAAGGAACTCGAAGATGCCCTC 4CL1^^P .TAEDA AGAGAGCGTTTTCCCAAGGCCATTTTCGGGCAGGGCTACGGCATGACAGAAGCAGGCCCG 4CL1_ P .MASSONIANA AGAGAGCGTTTTCCCAAGGCCATTTTCGGGCAGGGCTACGGCATGACAGAAGCAGGCCCG 4CL1_ P .TAEDA GTGCTGGCAATGAACCTAGCCTTCGCAAAGAATCCTTTCCCCGTCAAATCTGGCTCCTGC 4CL1_ P .MASSONIANA GTGCTGGCAATGAACCTAGCCTTCGCAAAGAATCCTTTCCCCGTCAAATCTGGCTCCTGC 4CL1_ P .TAEDA GGAACAGTCGTCCGGAACGCTCAAATAAAGATCCTCGATACAGAAACTGGCGAATCTCTC 4CL1_ P .MASSONIANA GGAACAGTCGTCCGGAACGCTCAAATAAAGATCCTCGATACAGAAACTGGCGAGTCTCTC 4CL1_ P .TAEDA CCGCACAATCAAGCCGGCGAAATCTGCATCCGCGGACCCGAAATAATGAAAGGATATATT 4CL1_ P .MASSONIANA CCGCACAATCAAGCCGGCGAAATCTGCATCCGTGGACCCGAAATAATGAAAGGATATATT 4CL1__P .TAEDA AACGACCCGGAATCCACGGCCGCTACAATCGATGAAGAAGGCTGGCTCCACACAGGCGAC 4CL1_ P .MASSONIANA AACGACCCGGAATCCACGGCCGCAACAATCGATGAAGAAGGCTGGCTCCACACAGGCGAC 4CL1_^P .TAEDA GTCGAATACATTGACGATGACGAAGAAATCTTCATAGTCGACAGAGTAAAGGAGATTATC 4CL1_ P .MASSONIANA GTCGGGTACATTGACGATGACGAAGAAATCTTCATAGTCGACAGAGTAAAGGAGATBATC 4CL1_ P .TAEDA AAATATAAGGGCTTCCAGGTGGCTCCTGCTGAGCTGGAAGCTTTACTTGTGGCTCATCCG 4CL1__P .MASSONIANA AAATATAAGGGCTTCCAGGTGGCTCCTGCTGAGCTGGAAGCTTTACTTGTGGCTCATCCG 4CL1_ P. .TAEDA TCAATCGCTGACGCAGCAGTCGTTCCTCAAAAGCACGAGGAGGCGGGCGAGGTTCCGGTG 4CL1_ P. .MASSONIANA TCAATCGCTGACGCAGCAGTCGTTCCTCAAAAGCACGAGGAGFTCGGGCGAGGTTCCGGTG 4CL1_ P..TAEDA GCCTTCGTGGTGAAGTCGTCGGAAATCAGCGAGCAGGAAATCAAGGAGTTCGTGGCAAAG 4CL1_ P..MASSONIANA GCGTTCGTGGTGAAGTCATCGGAAATCAGCGAGCAGGAAATCAAGGAGTTCGTGGCAAAG 4CL1__P..TAEDA CAGGTGATTTTCTACAAGAAAATACACAGAGTTTACTTTGTGGATGCGATTCCTAAGTCG 4CL1_ P.MASSONIANA CAGGTGGTTTTCTACAAGAAAATACACAGAGTTTACTTTGTGGATGCGATTCCGAAGTCG 4CL1_ P. TAEDA CCGTCCGGCAAGATTCTGAGAAAGGATTTGAGAAGCAGACTGGCAGCAAAATGA 4CL1 P.MASSONIANA CCGTCCGGCAAGATTCTGAGAAAGGATTTGAGAAGCAGACTGGCAGCAAAATGA Hinh 2. So sanh trinh tu nucleotide cua gen 4CL1 phan lap dugc tf Thong duoi ngga voi trinh tu nucleotide cua gen 4CL1 i tr cay Thong l r . 4CL1_P.massoniana: Trinh tg nucleotide cua gen 4CL1 tach tf Thong duoi ngua; 4CL1_P.taeda: Trinh i ia i tg nucleotide gen 4CL1 cua cay Thong l r (Ma so PTU12012). ia 482
Tgp chi Cdng nghe Sinh hgc 7(4): 479-483, 2009 KET LUAN Struture and evolution of 4-coumarate: Coenzyme A ligase (4CL) gene families. J Biol Chem 382: 645-654. Da phan lap thanh cong gen 4CL1 tir mRNA ciia Ehlting J, Shin JJK, Douglas CJ (2001) Identification of 4- cay Thong duoi ngua (Pinus massoniana Lamb.). coumarate: coenzyme A ligase (4CL) substrate recognition domains. Plant J 27: 455-465. Toan bg vimg khung dgc (ORF) mang ma di truyen cua gen 4CL1 a cay Thong duoi ngua gom 1614 Hamberger B, Hahlbrock K (2003) The 4-coumarate:CoA nucleotide ma hoa cho phan tu enzyme 4- ligase gene family in Arabidopsis thaliana comprises one coumarate:coenzyme A ligase gom 537 amino acid. rare, sinapateactivating and three commonly occurring Trinh tu nucleotide cua gen nay da dugc dang ky isoenzymes. Proc Natl Acad Sci USA 101(7): 2209-2214. tren Ngan hang gen (ma so FJ810495). Kumar A, Ellis BE (2003) 4-coumarate:CoA ligase gene family in Rubus idaeus: cDNA structures, evolution, and Loi cam on: Cdng trinh hodn thdnh voi su hd trg expression. Plant Mol Biol 51(3): 327-340. kinh phi cua di tdi thugc Chuang trinh Cdng nghe Lu H, Zeng QY, Zhao YL, Wang SS, Jiang XN (2003) sinh hgc Ndng nghiip vd Thiiy sdn, Bd Ndng nghiip Xylem-specific expression of a GRP1.8 promoter: 4CL vd Phdt triin ndng thdn. gene construct in transgenic tobacco. Plant Growth Regul 41(3): 279-286. TAI LIEU THAM KHAO Lu H, Zhao YL, Jiang XN (2004) Stable and specific expression of 4-coumarate:coenzyme A ligase gene Christensen JH, Baucher M, O'Connell AP, VanMontagu (4CL1) driven by the xylem-specific Pto4CLI promoter in M, Boetjan W (2000) Control of lignin biosynthesis. In the transgenic tobacco. Biotechnol Lett 26(14): 1147-1152. Molecular Biology of Woody Plants, Volume 1, ed. Jain SM, Minocha SC, For Sci 64: 227-267. Dordrecht: Zenn ZC, Yi CC, Yi HC, Yen L (2006) cDNA Cloning Kluwer. and Molecular Characterization of 4-Coumarate: Coenzyme A Ligase in Eucalyptus camaldulensis. Taiwan Cukovic D, Ehlting J, VanZiffle JA, Douglas CJ (2001) J For Sci 2l(\):%7-too. ISOLATION OF 4CL1 G E N E F R O M PINUS MASSONIANA L A M B . IN V I E T N A M Ha Van Huan'' *, Ho Van Giang', Nguyen Nhu Ngoc', Bui Van Thang', Nong Van Hai^, Chu Hoang Ha^ Vietnam Forestry University (VFU) Institute of Bioechnology (IBT) SUMMARY Lignin is a complex aromatic polymer of vascular plants that provides mechanical strength to the stem and protects cellulose fibres fi-om chemical and biological degradation. 4CL1 gene encoding 4-coumarate-CoA ligase, which play a key role in lignin biosynthesis pathway in plants. The fiiU-length cDNA of 4CL1 gene isolatedfiromPinus massoniana Lamb, contains 1614 bp encoding 537 amino acid residues. We report on the cloning and sequencing of 4CL1 gene from secondary developing xylem tissues oi Pinus massoniana Lamb. Comparison of nucleotide sequences of 4CL1 genefiromPinus taeda (Accession no., PTU12012) and Pinus massoniana Lamb, showed 98% similarity at nucleotide level and 99% at amino acid level. The nucleotide sequence of 4CL1 gene from Pinus massoniana Lamb, was deposited at NCBI database with the accession number FJ810495. Keywords: Ligin, Pinus massoniana Lamb., quality of wood, 4CL gene, 4-coumarate: coenzyme A ligase • Author for correspondence: Tel: 84-4-33724823; Fax: 84-4-33840063; E-mail: hvhuanbiotech(d),gmailcom 483

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