2010,32 (3) : ,283 http :/ / xuebao. jlau. edu. cn Journal of Jilin Agricultural University E2mail : sina. com Ξ 1ΞΞ, 1, 1, 2, 1 1.

Size: px

Start display at page:

Download "2010,32 (3) : ,283 http :/ / xuebao. jlau. edu. cn Journal of Jilin Agricultural University E2mail : sina. com Ξ 1ΞΞ, 1, 1, 2, 1 1."

那刁
4 years ago
Views:

1 2010,32 (3) : ,283 http :/ / xuebao. jlau. edu. cn Journal of Jilin Agricultural University E2mail : sina. com Ξ 1ΞΞ, 1, 1, 2, 1 1., ; 2., : GenBank (S73324),RT - PCR, bp cdna,genbank( : FJ786257, ), ppiczac :,,, N, ; Lys2277,Lys2277 : ; ; ; ; : Q78 : A : (2010) Cloning and Sequence Analysis of Alliinase Gene from Garlic and Con2 struction of Pichia pastoris Expression Plasmid WU Xiao2li 1, XU Jian 1, ZHANG Ting 1, J IA Ping 2, ZHOU Yun2kai 1 1. College of Life Science, Zhejiang Chinese Medical University, Hangzhou , China ; 2. Zhejiang Provincial People s Hospital, Hangzhou , China Abstract : Primers were designed based on the reported sequences of alliinase (S73324) in GenBank. The cdna gene encoding alliinase protein was extracted from Zhejiang garlic bulb by reverse transcription polymerase chain reaction (RT - PCR). PCR product was about bp DNA segment. The sequence was submitted to GenBank (accession number : FJ ), named Zhejiang alliinase ( ZJ alliinase). ZJ alliinase was compared with the other alliinases reported in aspect of homology and evolution,the protein structure and the enzyme active center were also predicted by bioinformatics softwares. The Alliinase gene was cloned to the ppiczac vector to construct the Pichia pastoris expression plasmid. The results showed the high similarity and approximation to those sequences of alliinase such as garlic, onion, scallion, alli2 um ascalonicum and chive,the target gene was successfully inserted to the ppiczac vector. The three di2 mensional structure of ZJ alliinase was arborization. A EGF2like domain was in the ZJ alliinase N2termi2 nal, and an aspartate aminotransferase (AAT) superfamily domain was in its center. The amino acid residue Lys2277 could be the combining site of pyridoxal phosphate, and the sequence around it could be the potential enzyme active center. The ppiczac2alliinase recombination plasmid was completely accurate by using restrictional enzyme assay, plasmid PCR identification and recombinant plasmid sequence analy2 Ξ ΞΞ : ( Y ), (2009ZZ05) :,,,, : :

2 : 259 sis. Key words : alliinase ; garlic ; cloning ; sequence analysis ; Pichia pastoris (Allicin),, [1 ], (Alliinase),,,, [2 ], DH5 ppiczac,p GEMP2 T Easy Invitrogen 113 Trizol PCR Invitrogen Reverse Transcriptase XL PrimeStar HS DNA T 4 TakaRa DNA TakaRa GenBank 1 5 2GGTATAAAT2 GATCTGCCTAGTGATT23, 5 2CATAC AAAGATACTCCTTACTGCC23, Invitrogen RNA RT - PCR,Trizol, Trizol RNA, cdna, PCR 94 5 min ;98 10 min, min, 72 3 min, 30 ; 72 5 min 1 % PCR p GEMP2T2 PCR PGEMP2T Easy, p GEMP2T2 DH5, 37,,1 % EcoR, TakaRa 115 GenBank Gen2 Bank, ( ZheJiang alliinase, ZJ alliinase) DNAstar DNASYS 116 p GEMP2T2, 5 2CCCTCGAGAAGAGAGA2 GATGATCTGCCTAGTG23, 5 2ATAA2 GAATGCGGCCGCAATGAAAGGACGAC23 Xho Not, PCR PCR ppiczac T 4 DNA,pPICZaC2Al2 liinase PCR, RT - PCR p GEMP2T2 RT - PCR 1 %, bp ( 1) p GEMP2T bp, EcoR,3 015 bp bp ( 2), 212, DNAStar, bp,orf,0rf Journal of Jilin Agricultural University

2 60 2010 6 6 1 430 bp, 1 425 bp, ATG TAA GenBank( : FJ786257) 5413 kd, 7172, 474, 55 () 53 ( ) 150 145,, 1116 %, Lys,6118 %, [3 ] 24150 % 172,217,354),Aaron Rabinkov [4

4 N2 ( Gly61,143, 155,164), 1. RT - PCR RT - PCR product ; 2. Negative control ;M. DL 2000 Marker 1 RT - PCR Fig. 1. RT - PCR products of alliina se 3

3 bp, bp, ATG TAA GenBank( : FJ786257) 5413 kd, 7172, 474, 55 () 53 ( ) ,, 1116 %, Lys,6118 %, [3 ] % 172,217,354),Aaron Rabinkov [4 ] 4,, Asn19 Asn146 Asn191 Asn328 1 cgmp (Lys 451) 11 c ( Thr31,134,311, 332,336 ;Ser62,77,319,362,422,469) 8 ( Thr72, 134, 311, 336, 433 ; Ser219,248, 366) 2 (Lys199,247) 4 N2 ( Gly61,143, 155,164), 1. RT - PCR RT - PCR product ; 2. Negative control ;M. DL 2000 Marker 1 RT - PCR Fig. 1. RT - PCR products of alliina se 3 Fig. 3. Secondary structure prediction of alliina se 214, p GEM2T2Alliinase EcoR Products of p GEM2T2Alliinase recombinant plasmid digested with EcoR ; M. 250 bp DNA Ladder Marker 2 p GEMP2T2EcoR Fig. 2. Analysis of p GEM2T2alliina se recombinant pla s2 mid dige sted with EcoR 213, (2HOX - D) % 3, %, [2 ] 27 % 30 % ; %,5 (Asn19,45, 4 Fig. 4. Three dimensional structure N 1 ( 5), , C - X C - X - C - X 2 - C - X 5 - C - X 6 - C,,,, [5 ] Journal of Jilin Agricultural University 2010, J une

: 261 ( 6),,147 310, 336 474 5 2 ( PLP), 5 Fig. 5. Alliina se EGF2like domain 6 Fig. 6. Aspartate aminotransfera se superfamily domain 215 5 2 ( PLP), 285 [6 ],Lys2280 Lys2280 C - S,280 285 Lys [7 ] Manabe T [7 ] C - S, ( Allium cepa L.

4 : 261 ( 6),, , ( PLP), 5 Fig. 5. Alliina se EGF2like domain 6 Fig. 6. Aspartate aminotransfera se superfamily domain ( PLP), 285 [6 ],Lys2280 Lys2280 C - S, Lys [7 ] Manabe T [7 ] C - S, ( Allium cepa L. ) ( Allium tuberosum L. ) ( Allium sativum L. ) ( A. sativum) ( A. sativum ) ( Ara2 bidopsis thaliana) 2 ( A. thaliana) 2 ( Saccha2 romyces cerevisiae) 2 2 ( S. cerevisiae) 2 2 ( S. cerevisi2 ae) 2 ( Pseudomonas aeruginosa) O2 11 Lys2280 ( 7) Lys2277 C - S,Lys2277,Lys2277, [8 ], : 1 ; The numbers in paren2 theses indicate the location of the first amino acid of the region in the respective complete sequence,the Kresidue indicated by an arrow is the binding site of Pxy2P 7 Fig. 7. Prediction of alliina se active center 216, 9815 % DNASYS (M98267) 8716 %, (AF409947) GenBank 9013 %, (AF409950) (gi : S73324) : 8918 %, ( AF ) 3 GTA, %, Journal of Jilin Agricultural University

5 , 6214 %, 4012 % 5514 %,,Rabinkov [9 ] ( 8),,,,, 217 ppiczac bp, PCR 8 Fig. 8. Evolution analysis of alliina s bp,ppiczac2alliinase bp ( 9) Xho Not bp bp 2 ( 10) PCR, bp ( 11),, 1 3. ppiczac2alliinase Xho / EcoR Products of ppiczac2alliinase recombinant plasmid digested with Xho / EcoR ; M. 250 bp DNA Ladder Marker 10 p PICZaC2Alliina se Fig. 10. Analysis of p PICZaC2Alliina se pla smid dige sted recombinant 1 3. ppiczac2alliinase ppiczac2alliinase recombi2 nant plasmid ;M. 250 bp DNA Ladder Maker 9 p PICZaC2Alliina se Fig. 9. p PICZaC2Alliina se recombinant pla smid 1 3. PCR PCR products ; M. 250 bp DNA Ladder Marker 11 p PICZaC2Alliina se PCR Fig. 11. PCR products of p PICZaC2Alliina se recombi2 nant pla smid Journal of Jilin Agricultural University 2010, J une

6 : ( S22L2, EC ), 10 % 12 % [10 ] D, D [11 ],,,, [12 ],,,,,,,,,,, [13214 ],,, [15 ],,,, GenBank,RT - PCR GenBank 3, 9 RT - PCR DNA PrimeStar HS DNA,,, DNA GenBank, 9815 %, 5 2,,, 2,,,,,, ;, ;, ;,, [16 ], : [1 ],. [J ]., 2004,11 (6) : [2 ],,,. [J ].,2004,40 (3) :356. [3 ],. [J ].,2006,27 (10) : [4 ] Rabinkov A, Wilchek M, Mirelmand D. Alliinase (alliin lyase) from garlic ( Alliium sativum) is glycosylated at ASN 146 and forms a complex with a garlic mannose2specific lectin [J ]. Glycoconjugate Journa,1995 (12) : [5 ] Kuettner E B, Rolf H, Manfred S W. Purification, characteriza2 tion, and crystallization of alliinase from garlic[j ]. Archives of Bio2 chemistry and Biophysics,2002 (402) : [6 ] Lancaster J E,Shaw M L, Randle W M. Differential hydrolysis of Alk ( en) ly cysteine suphoxides by alliinase in onion macerates : Flaour implications[j ]. J Sci Food Agric,1998,78 : [ 7 ] Manabe T, Hasumi A,Sugiyame M, et al. Alliinase [ S2alk(en) yl2 L2cysteine sulfoxide lyase ] from Allium tuberosum (Chinese chive) Purification, localization, cdna cloning and heterologous function2 al expression[j ]. Eur J Biochem,1998, 257 : ( 283 ) Journal of Jilin Agricultural University

7 : 283,TDZ,,NAA,, [223 ] [4 ],,, [5 ] 62BA IBA 30 g/ L,,(20 g/ L) (40 g/ L), [6 ],,,, AgNO 3, [728 ] 2 mg/ L AgNO 3 : MS + 62BA 410 mg/ L + IBA 0115 mg/ L + 30 g/ L,, 5 d : [1 ],. [J ].,2006 (3) : [2 ],. [J ].,2004,31 (5) : [3 ],. [J ].,2006,28 (4) : [4 ],. [J ].,2005,27 (4) : [5 ] BU X X, CHEN W L. The effect of activated charcoal on the ab2 sorption of plant growth regulators in culture medium [ J ]. Acta Phytophysiologica Sinica,1988,14 (4) : [6 ],,. [J ].,2007,34 (6) : [7 ],,. AgNO 3 [J ].,1997,11 (1) : [8 ],. [J ].,2002,25 (3) : ( 263 ) [8 ],,. [J ].,2004 (9) : [9 ] Rabinkov A, Zhu X Z, Grafi G,et al. Alliin lyase (Alliinase) from garlic ( Allium sativum) Biochemical characterization and cd2 NA cloning [J ]. Appl Biochem Biotechnol, 1994, 48 ( 3) : [10 ],. [J ].,2007,28 (5) : [11 ],. [J ].,2003, 31 (6) : ,1051. [12 ] Kuettner E B, Hilgenfeld R, Weiss M S. Purification, characteri2 zation, and crystallization of alliinase from garlic [ J ]. Arch Biochem Biophy, 2002,402 : [13 ],. [J ].,2008,17 (2) : [14 ],,,. [J ].,2005, 22 (5) : [15 ]. [J ].,2004,39 (8) :9210. [16 ],,,. [J ].,2006,17 (4) : ( 267 ) [10 ],,,. 14 [J ].,2007,22 (2) : [11 ] Goodwin T W. The biochemistry of Carotenoids[J ]. Plant,Chap2 man and Hall,1980 (1) :529. [12 ],. [J ]., 1992 (3) :19. [13 ],,. [J ].,1997 (1) :628. [14 ],,,. [J ].,2007 (1) :426. [15 ], 1 [M]. :,2008. [16 ]. [ M]. :,2000. [17 ],,,. [J ].,2005,28 (1) : [18 ],. [J ].,1998,7 (3) : [19 ],. [J ].,2006,30 (3) : [20 ]. 6 [D]. :,2009 : Journal of Jilin Agricultural University

标题

吉林农业大学学报２０１６３８１８７９１ＪｏｕｒｎａｌｏｆＪｉｌｉｎＡｇｒｉｃｕｌｔｕｒａｌＵｎｉｖｅｒｓｉｔｙｈｔｔｐｘｕｅｂａｏｊｌａｕｅｄｕｃｎＥｍａｉｌｊｌｎｄｘｂｖｉｐｓｉｎａｃｏｍ神经生长因子ｍｒｎａ在鹅等级前卵泡中的表达刘璐璐隋玉健朱海洋武惠岩杨辉孙永峰吉林农业大学动物科学技术学院长春１３０１１８