44 3 2 0 0 8 3 SCIENTIA SILVAE SINICAE Vol144,No13 Mar.,2 0 0 8 FAD2 cdna ( 410004) : FAD2, EST, 5 RACE PCR FAD2 cdna 1 682 bp, 1 149 bp, 382, FAD2, FAD2 FAD2 : ; EST ; FAD2 ; RACE; PCR ; :S718146 ;Q94312 :A :1001-7488 (2008) 03-0070 - 06 Cloning of Full2Length cd NA of FAD2 Gene from Camellia oleifera Tan Xiaofeng Chen Hongpeng Zhang Dangquan Zeng Yanling Li Wei Jiang Yao Xie Lushan Hu Xiaoyi Hu Fangming ( Key Laboratory of Non2Wood Forest Product of State Forestry Administration Central South University of Forestry and Technology Changsha 410004) Abstract : Camellia Oleifera is an important oil2producing woody species in China, and the oil, called as tea2oil, can be used as a high2quality edible oil, cosmetic material and biomass energy with the abundant poly unsaturated fatty acids such as oleic acid and linoleic acid. FAD2 gene is a key factor in controlling lipid biosynthesis, and controls the formation of linoleic acid from oleic acid in seeds of C. oleifera. Cloning of the FAD2 gene would facilitate us to reveal the lipid synthesis rule in seeds of C. oleifera and have important impact on theory and application. Full2length cdna clone of FAD2 gene was for the first time obtained by using methods of 5 RACE and overlap extension PCR based on our constructed EST library of C. oleifera. It was 1 682 bp in length and contained a 1 149 bp ORF (open reading frame) encoding a peptide of 382 amino acids, which had the typical conserved domains of FAD2 and showed high homology with those of other plant species. Key words : Camellia oleifera ; EST library ; FAD2 gene ; RACE; Overlap Extension PCR ; gene cloning ( Camellia oleifera),, (,2006 ;,2005),,, SAD ; FAD, FAD2 12 13,, FAD2 2004, 1 4 cdna (,2004 ;,2005), EST (,2006), 1 FAD2, cdna, cdna, 5 RACE (Chenchik et al., 1998) PCR FAD2 cdna 1 111 2006 9, 1, - 75 cdna, EST :2007-09 - 24 : (2006BAD18B0204, 2006BAD01A1706), ( 30371184), (07JJ3070), (06FJ4111)
3 : FAD2 cdna 71 DH5 pmd182t Total RNA Purification System SMART RACE Kit Puprep Gel Extraction Kit TaKaRa Invitrogen Clontech Ambiogen,DEPC H 2 O 2 Tris EDTA EB - 112 11211 cdna FAD2 cdna FAD2, T3 T7, Vector NTI 910, BLAST X 11212 RNA 1,,,, Total RNA Purification System RNA 11213 RACE FAD2 cdna, Primer Premier 510 1 GSP :5 - GTGGTGACGGCGGTGACTGTATTTC - 3 ( ) SMART2 RACE kit Puprep Gel Extraction Kit RACE, TA 5 RACE pmd182t, DH5 X2gal IPTG LB, LB, DNA,PCR, 11214 PCR FAD2 cdna EST RACE FAD2 cdna, PCR OE2F1 OE2R1,OE2F2 OE2R2, OE2F1 OE2R2 cdna,oe2r1 OE2F2 5 RACE FAD2 cdna, 13 bp,, : OE2F1 : 5 - TCTAATACGACTCACTATAGGGCAAG - 3 ; OE2R1 : 5 - ACACAACCTTGAAGTGCCCAG - 3 ; OE2F2 : 5 - TTCAAGGTTGTGTCCTCACTGGT - 3 ;OE2R2 : 5 - CTACATGTTCATTACATACCAAAGCC - 3 OE2F1 OE2R1 5 RACE DNA,OE2F2 OE2R2 FAD2 DNA, T1 T2,2 1 :94 3 min ; 94 30 s,68 30 s,72 3 min,35 ; 72 10 min ; 4 PCR, T1 T2 200 L 110 L Pfu,610 L buffer, 610 L dntp Mix,1010 L T1,1010 L T2,2110 L, PCR :94 5 min ; 94 45 s,55 1 min,72 70 s,5 ; 72, 10 min ; 4 5 min,, 310 L OE2F1,310 L OE2R2,, PCR, :94 5 min ; 94 45 s,60 45 s,72 2 min,35 ; 72 5 min ; 4 PCR DNA PCR, 1 T1 T2 PCR Tab. 1 PCR reaction systems of T1 and T2 T1 PCR reaction system of T1 T2 PCR reaction system of T2 Pfu 015 L Pfu 015 L 10 Pfu buffer 5 L 10 Pfu buffer 5 L dntps 4 L dntps 4 L OE2F1 Primer OE2F1 015 L OE2F2 Primer OE2F2 015 L OE2R1 Primer OE2R1 015 L OE2R2 Primer OE2R2 015 L 5 RACE DNA Recombinant plasmid DNA of 5 RACE Ultra2pure water Total 115 L FAD2 DNA Recombinant plasmid DNA of FAD2 in cdna library 115 L 38 L Ultra2pure water 38 L 50 L Total 50 L 2 211 5 RACE RACE,, 1,DNA,
72 44 640 bp,, RACE RACE, DNA, PCR 2 2, 6 PCR 640 bp RACE DNA, 6 1, 636 bp, cdna 190 bp, 5 203 bp 433 bp, N 144 BLAST, FAD2 85 %, FAD2 5 1 5 RACE Fig. 1 Electrophoresis pattern of 5 RACE product 2 PCR Fig. 2 Identification of recombinants by PCR 212 PCR PCR T1 T2 ( 3) 3,T1, 500 bp,t2 1 200 bp 1, PCR T1 T2, FAD2 cdna, 4 4, PCR 1 700 bp, T1 T2, FAD2 cdna 3 T1 T2 Fig. 3 The electrophoresis result of T1 and T2 4 PCR Fig. 4 The electrophoresis result of Overlap Extension PCR 213 FAD2 cdna 5 RACE, 1 1 682 bp cdna, PCR, cdna 5, cdna 1 1 149 bp, 382,5 203 bp,3 311 bp, 1 19 bp polya FAD2 3 (Los et al., 1998 ; Shanklin et al.,1998) FAD2, 3 HECGHH HRHH HVAHH, 104 109 140 144 316 320, 5 1 2 30,, 2 3, 12 - FAD2, cdna 12 - FAD2 NCBI Blast X, 2 2,
3 : FAD2 cdna 73 5 FAD2 cdna Fig. 5 cdna and deduced protein sequences of FAD2 gene from Camellia oleifera ATG,TGA,OE2F1 OE2R1 OE2F2 OE2R2 PCR PCR, 5 RACE FAD2 cdna,3 ATG is start codon, TGA is stop codon,oe2f1, OE2R1, OE2F2 and OE2R2 are the primers for Overlap Extension PCR. Four arrows mark OE primers, the part underlined is the overlap of RACE product with FAD2 cdna in the library, and three His clusters are marked by shadow. FAD2 FAD2, 88 %, ( Sesamum indicum) 87 %, FAD2 FAD2, FAD2, 3,,,, 2, FAD2 ( 6) 3 CLONTECH Touchdown PCR RACE RACE GC (50 % 70 %) T m (70 ), Touchdown PCR UPM
74 44 GSP PCR,, NUP NGSP,, UPM GSP, NUP NGSP PCR PCR, Horton (1989),, PCR, DNA PCR, 2 FAD2 cdna Blast X Tab. 2 Blast X result of full2length cdna of FAD2 from C. oleifera Species Gene names Simelarity Sesamum indicum 62FAD2 332Π378 (87 %) Olea europaea 122FAD2 329Π378 (87 %) Ricinus communis 122FAD2 325Π378 (85 %) Punica granatum 122FAD2 316Π358 (88 %) Vernicia fordii 122FAD2 325Π376 (86 %) Solanum commersonii 122FAD2 320Π377 (84 %) Hevea brasiliensis 62FAD2 326Π378 (86 %) Vernicia montana 122FAD2 324Π376 (86 %) Cucurbita pepo 62FAD2 313Π361 (86 %) Gossypium hirsutum 122FAD2 329Π378 (87 %) Jatropha curcas 122FAD2 323Π379 (85 %) Nicotiana tabacum 62FAD2 315Π360 (87 %) Glycine max 62FAD2 315Π377 (83 %) Tropaeolum majus 122FAD2 316Π377 (83 %) Linum usitatissimum 122FAD2 320Π376 (85 %) 6 FAD2 Fig. 6 Phylogenetic tree based on FAD2 of C. oleifera and other species 2, OE2R1 OE2F2 13 bp, T1 T2 13 bp, T1 T2 FAD2 cdna cdna, FAD2, 5 RACE, FAD2 cdna 5 cdna, PCR 5 RACE cdna FAD2 cdna, FAD2 FAD2, FAD2, FAD2,,,
3 : FAD2 cdna 75,,,. 20061. :,370-3831,,,. 20041 cdna.,24 (5) :1-51,,. 20051., (6) :7-91,,,. 20051., 25 (4) :17-231,,,. 20061 EST.,42 (1) :43-481 Chenchik A, Zhu Y Y, Diatchenko L, et al. 19981 Generation and use of high2quality cdna from small amounts of total RNA by SMART PCR. BioTechniques Books,MA :305-3191 Horton R M, Hunt H D, Ho S N, et al. 19891 Enginnering hybrid gene without the use of restriction enzymes : gene splicing by overlap extention. Gene, 77 (1) :61-68. Los D A, Murata N. 19981 Structure and expression of fatty acid desaturases. Biochim Biophys Acta,1394 : 3-151 Shanklin J, Cahoon E B. 19981Desaturation and related modifications of fatty acids. Ann Rev Plant Physiol Plant Mol Biol, 49 (6) :611-6411 ( ) (,,2008),, 2 ; 5,, 5,,2005,2007,,,, ( ) : 43. 00 ; 53. 00 :350002 :