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戌温刁
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1 作物学报 ACTA AGRONOMICA SINICA 200, 36(5): ISSN ; CODEN TSHPA9 DOI: /SP.J 芥菜型油菜 FAE 基因序列特征及其与芥酸含量关系的初步分析徐爱遐,2 黄镇 2 马朝芝肖恩时 2 张修森 2 涂金星,* 傅廷栋, ; 2, 7200 : 6 (Brassica juncea)( ) 2 ( ) FAE, 9 FAE 522 bp,, 507, FAE (BjFAE. BjFAE.2), FAE (BrFAE BnFAE), BjFAE. BrFAE, BjFAE.2 BnFAE; BjFAE. BjFAE.2 7 bp Hind III ( ), 5 FAE, BjFAE. 2 SNP ( ), BjFAE.2 2 SNP ( ), 4 SNP 3 ( ) BjFAE. 968 (C T) 323 (Thr Ile), ( ) ; 265 (T C) 422 (Phe Ser), ( ), BjFAE.2 49 (T C) 7 (Phe Leu), ( ) FAE, 265 : ; FAE; ; FAE Sequence Characteristics and Its Relationship with Erucic Acid Content in Brassica juncea XU Ai-Xia,2, HUANG Zhen 2, MA Chao-Zhi, XIAO En-Shi 2, ZHANG Xiu-Sen 2, TU Jin-Xing,*, and FU Ting-Dong National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan , China; 2 College of Agriculture, Northwest A&F University, Yangling 7200, China Abstract: Low erucic acid content, which may result from the differential mutation of the fatty acid elongation (FAE) gene, is a major breeding target in canola quality improvement. We cloned the FAE genes in six Brassica juncea varieties (with high, intermediate and low contents of erucic acid), and their sibling species: two B. rapa (with high and low erucic acid contents) and one B. nigra varieties by using the method of homologous sequencing. The results showed that the sequences of FAE genes in nine varieties were 522 bp without introns, and all encoded a protein of 507 amino acids. There were two FAE gene sequences (BjFAEBjFAE. and BjFAE.2) in B. juncea, and only one FAE gene sequence was in B. rapa (BrFAE) and B. nigra (BnFAE), respectively. BjFAE. was corresponding to the BrFAE of B. rapa; and BjFAE.2 was corresponding to BnFAE of B. nigra. There were 7 nucleotide variations and the different Hind III restriction sites (No. 45 and No. 44) and 5 amino acid variations in protein construction between BjFAE. and BjFAE.2. The FAE gene sequences comparison analysis showed that there were two SNPs (No. 968 and No. 265) in BjFAE., two SNPs (No. 49 and No. 237) in BjFAE.2, three of the four SNPs (No. 49, No. 968, and No. 265) resulted in differences in the amino acid level. The No. 323 amino acid in BjFAE. gene was (2005C08), (863 )(2009AA005) (A ) * (Corresponding author):, tujx@mail.hzau.edu.cn; Tel: : xuaixia64@yahoo.com.cn; Tel: Received(): ; Accepted( :

2 5 : FAE 795 changed (Thr Ile) duo to the change of the No. 968 base (C T)), which could explain the decrease of erucic acid content in B. juncea and B. rapa. The point mutation at the No. 265 base (T C) resulted in the change at No. 422 amino acid (Phe Ser), which could partially explained the variations from high erucic acid to low erucic acid in Brassica juncea, but no difference was found in No. 265 in B. rapa varieties with high and low erucic acid content. The mutation at No. 49 base (T C) resulted in No.7 amino acid change (Phe Leu), which could explained the variations from intermediate erucic acid to high erucic acid (erucic acid) in B. juncea. We compared the FAE gene sequences of the low erucic acid yellow mustard mutant called from northern Shaanxi and the varieties with low erucic acid from foreign countries. The results showed that their difference was only in No.265 base. Keywords: Brassica juncea; FAE; Cloning; Sequence analysis (C22:),,,,,,, 25% [], ( 40%) %(278-3), FAE(Fatty Acid Elongation ) [2-3] James [4] (Arabidopsis) FAE ( U2942),, Clemens [5] FAE FAE cdna Barret [6] FAE, cdna, FAE cdna (CE7 CE8),, CE7 E, CE8 E2 ; Fourmann [7] FAE (BnFAE. BnFAE.2), BnFAE. BnFAE.2 QTL (E E2), BnFAE. BrFAE( FAE ), BnFAE.2 BO-FAE( FAE ) Han [8], (Askari) (Drakkar) FAE 507, 282, (Ser), (Phe) [9] [0] FAE, 9 FAE 4, Gupta [] FAE. FAE.2, FAE. 4 SNP (single nucleotide polymorphism), FAE.2 3 SNP, FAE FAE, FAE, SNPs, 材料与方法. 6 ( ),,, 2, 2, ( ) P2(4923), DNA,.2 DNA Doyle [2] CTAB DNA.0% DNA, (Pharmacia Biotech, GeneQuant II) DNA, DNA 50 ng μl, 20.3 PCR P P2 3 DNA(P- P-2 P-3 P2- P2-2 P2-3) PCR,

3 Code Material Table Genome 表供试材料 Materials used in the experiments Erucic acid content (%) Source P 278 Mutant 278 AABB <.0 Shaanxi, China Spring seeding P AABB <.0 Australia Spring seeding P3 Wuqi mustard AABB 25.6 Shaanxi, China Spring seeding P4 Yan anxiaping mustard AABB 23.8 Shaanxi, China Spring seeding P5 Chang an mustard AABB 45.4 Shaanxi, China Winter seeding P6 Baoji mustard AABB 46.7 Shaanxi, China Winter seeding P7 Nigra BB >40.0 Hubei, China Spring seeding P8 558 Rapa 558 AA <.0 Gansu, China Winter seeding P9 Yellow rapa AA 46.3 Gansu, China Winter seeding Type 7 ( ) 5 DNA (P3 P4 P5 P6 P7 P8 P9) PCR PCR, pgem-t vector, 3 DNA FAE GenBank FAE ( AJ55897, 522 bp) GF/GR (5 -ATGACGTCCAT TAACGTAAAGCTCC-3 5 -ATTAGGACCGACC GTTTTGGACA-3 ), 9 3 DNA, FAE PCR : 50 ng DNA, 50 ng (GF GR), PCR, U Taq, 0.5 mmol L dntps 2.0 mmol L Mg 2+, ddh 2 O 20 μl PCR 94 3 min, s, s, 0.5, 72 min, 3 ; 94 min, s, 72 min, 25 ; 72 5 min, 0 PCR.0%.4 FAE UNIQ0 Column DNA Collection Kit, pgem-t Easy vector (Promega, USA) 3~5, NCBI Blast,.5 DNA DNAStar FAE,, Clustal W ( ac.uk/clustalw/) 2 结果与分析 2. FAE GF/GR 3 DNA,.0%, 500~ 600 bp ( ), 3 DNA, 3~5 GenBank FAE, GF/GR 3 DNA FAE, 522 bp, FAE, 3 FAE 图引物 GF/GR 在 3 个 DNA 样品中的扩增结果 Fig. Amplification patterns of primer combination GF/GR in 3 DNA samples M: 00 bp maker; P~P9: M: 00 bp DNA ladder; P P9 compared with the codes given in Table.

4 5 : FAE FAE 3 FAE FAE, FAE FAE (U2942) 88.4%, FAE 95%, AJ55897 (FAE.) AJ55898 (FAE.2) ( 99%), GenBank AF49876 (FAE.) AF49877 (FAE.2) (97%) P~P6 FAE BjFAE. BjFAE.2, (P8 P9) (P7) (BrFAE BnFAE),, BjFAE. BrFAE, BjFAE.2 BnFAE, FAE (BjFAE. BjFAE.2) FAE (BrFAE BnFAE) FAE GenBank 3 FAE (AF49046 AF400049, AJ55897 AJ55898), 2, BjFAE.2 AJ55898 (BjFAE.2) BnFAE, AF (B. nigra); BjFAE. BrFAE (high erucic), BrFAE (low erucic) AF49046(B. rape) AJ55897(BjFAE.) FAE BjFAE. BjFAE.2 A B, BjFAE. BjFAE.2 7 ( 2), 22, 49 ; BjFAE. 45 Hind III, BjFAE.2 Hind III 44 图 2 白菜型油菜黑芥和芥菜型油菜 FAE 基因系谱分析 Fig. 2 Genealogical analysis of FAE in B. rapa, B. nigra, and B. juncea 表 2 芥菜型油菜 BjFAE. 和 BjFAE.2 的核苷酸差异位点 Table 2 Positions of SNP between BjFAE. and BjFAE.2 in B. juncea Single nucleotide variation sites (bp) Gene BjFAE. C T A C T C C A C C C T T T A BjFAE.2 C/T C C C/T C A G G T A T C A C T BjFAE. G A C T A G T C T C C T A A T BjFAE.2 A G A C T A G T C T T C G T C BjFAE. A T C G C T C/T G C T G G T C G BjFAE.2 G C A A A A C A T C A T C A C BjFAE. G T A C A T A A C T A C T/C A A BjFAE.2 A C G T G C G G T C C G T T G BjFAE. G C A A A C C T G A C BjFAE.2 A G G G G T T A C G T

5 FAE SNP BjFAE ; BjFAE ( 2); BjFAE. 968, P- P-2 P-3 P2- P2-2 P2-3 P3 P4 T, P5 P6 C; BjFAE. 265, P- P-2 P-3 P2- P2-2 P2-3 C ( T), P3 P4 C, P5 P6 T ( 3) 968 (C T), 265 (T C)BjFAE. (A ) BrFAE, 968, P8 T, P9 C, BjFAE. ( 3), 968 SNP BjFAE.2 49, P- P-2 P-3 P2- P2-2 P2-3 P5 P6 C, P3 P4 T;, BjFAE.2 237, P- P-2 P-3 P2- P2-2 P2-3 P5 P6 T, P3 P4 C ( 3), SNP BjFAE.2 (B ) BnFAE, P3 P4, P- P-2 P-3 P2- P2-2 P2-3 P5 P6 ( 3) (P- P-2 P-3) FAE 4923 (P2- P2-2 P2-3), 9, BjFAE. 265, P- P-2 P-3 C, P2- P2-2 P2-3 C T ( 3) 2.4 FAE FAE 507, FAE, BjFAE. BjFAE.2, 7 5 (), , ( ) FAE, BjFAE , (P P2)(P3 P4) (Ile), (P5 P6) Table 3 Name 表 3 不同芥酸含量的芥菜型油菜的 BjFAE. 和 BjFAE.2 基因在第 49 第 237 第 968 和第 265 位碱基表现 Haplotype profiles of No. 49, No. 237, No. 968, and No. 265 base pairs of FAE. and FAE.2 genes in B. juncea varieties with variable erucic acid content FAE. (SNPs) FAE.2 (SNPs) Genome C22: (%) 49 bp 237 bp 968 bp 265 bp 49 bp 237 bp 968 bp 265 bp P- AABB < C C T C C T C T P-2 AABB < C C T C C T C T P-3 AABB < C C T C C T C T P2- AABB < C C T T C T C T P2-2 AABB < C C T C C T C T P2-3 AABB < C C T C C T C T P3 AABB 25 C C T C T C C T P4 AABB 25 C C T C T C C T P5 AABB >45 C C C T C T C T P6 AABB >45 C C C T C T C T P7 BB >45 T C C T P8 AA < C C T T P9 AA >45 C C C T

6 5 : FAE 799 (Thr); 422, (P5 P6) (Phe), (P3 P4) (Ser), (P P2) (Ser) , BjFAE.2 7, (P3 P4) (Phe), (P P2) (P5 P6) (Leu), 49, 49 BrFAE, (P8) 323 (Ile), (P9)(Thr), BjFAE., 968 (C T) 323 (Thr Ile), 3 讨论 (AABB)(AA) (BB),, BjFAE. BrFAE, BnFAE.2 BnFAE, FAE A B, Fourmann [7] FAE BjFAE. BrFAE BnFAE.2 BnFAE,, FAE BjFAE. BjFAE.2 GenBank AF49876(FAE.) AF49877(FAE.2 ), Gupta [] GenBank AJ55897(FAE.) AJ55898 (FAE.2), Gupta AF55897(FAE.) AF55898 (FAE.2) 67, BjFA E. BjFAE.2 7 Gupta [] BJFAE. 4 SNP ( ), BJFAE.2 3 SNP ( ), BjFAE. BjFAE.2 2 SNP ( ) ( 4) 3 ( ), , Table 4 Gene 表 4 比较不同研究者发现的芥菜型油菜不同芥酸含量 BjFAE. 和 BjFAE.2 中存在的 SNP SNPs comparisons in BjFAE. and BjFAE.2 of different erucic acid content from different researchers SNP SNP sites Erucic acid content 49 bp 237 bp 59 bp 735 bp 968 bp 265 bp 476 bp GU-FAE. High C C G C C T T Low C C A T T C T BjFAE. High C C A T C T T Intermediate C C A T T C T Low C C A T T C/T T GU-FAE.2 High T C T T C T T GU-FAE.2 Low C T T T C T A BjFAE.2 High C T T T C T A BjFAE.2 Intermediate T C T T C T A Low C T T T C T A 968 Gupta [], BjFAE. 968 (C T) ( ) [6-7,], BjFAE. E, 968 (C T)

7 E e BjFAE. 265 (T C), ( 4), 265 (C T) BjFAE Gupta Gupta, (E2) (e2),,, ( 4), BjFAE.2 E2 e2 SNP,, BjFAE.2 E2 FAE 968, BjFAE., ,, 265,,, 968 FAE., ; FAE, BjFAE., , Han [8] FAE. 282, [0] FAE 4, FAE. A,, BjFAE.2 7, 4 结论 FAE, BjFAE. BjFAE.2 FAE, FAE, 7 Hind III , BJFAE. ; , BJFAE , BjFAE. 968 (C T) ( ) BJFAE.2 49 (C T) References [] Xu A-X( ), Huang J-Y( ). Analysis and evaluation of rapeseed germplasm resources in Shaanxi province. J Northwest Agric (), 999, 8(3): (in Chinese with English abstract) [2] Lemieux B, Miquel M, Somerville C, Browse J. Mutants of Arabidopsis with alterations in seed lipid fatty acid composition. Theor Appl Genet, 990, 80: [3] Kunst L, Taylor D C, Underhill E W. Fatty acid elongation in developing seeds of Arabidopsis thaliana. Plant Physiol Biochem, 992, 30: [4] James D W, Lim E, Keller J, Plooy I, Ralston E, Dooner H K. Directed tagging of the Arabidopsis fatty acid elongation (FAE) gene with the maize transposon activator. Plant Cell, 995, 7: [5] Clemens S, Kunst L. Isolation of Brassica napus cdna encoding β-ketoacyl-coa synthase, a condensing enzyme involved in the biosynthesis of very long chain fatty acids in seeds. Plant Physiol, 997, 5: [6] Barret P, Delourme R, Renard M, Domergue F, Lessire R, Delseny M, Roscoe T J. A rapeseed FAE gene is linked to the E locus associated with variation in the content of erucic acid. Theor Appl Genet, 998, 96: [7] Fourmann M, Barret P, Renard M, Pelletier G, Delourme R, Brunel D. The two genes homologous to Arabidopsis FAE cosegregate with the two loci governing erucic acid content in Brassica napus. Theor Appl Genet, 998, 96: [8] Han J, Lühs W, Sonntag K, Zähringer U, Borchardt D S, Wolter F P, Heinz E, Frentzen M. Functional characterization of β-ketoacyl-coa synthase genes from Brassica napus L. Plant Mol Biol, 200, 46: [9] Xiao L(), Lu C-M( ). Cloning of fae gene partial sequence and SNP analysis in Brassica species. Sci Agric Sin ( ), 2005, 38(5): (in Chinese with English abstract) [0] Wu Y-H( ), Xiao L(), Wu G(), Lu C-M( ). Cloning of FAE gene partial sequence and molecular analysis of A/C genomes in Brassica. Sci China (), 2007, 37(): 35 4 (in Chinese) [] Gupta V, Mukhopadhyay A, Arumugam N, Sodhi Y S, Pental D, Pradhan A K. Molecular tagging of erucic acid trait in oilseed mustard (Brassica juncea) by QTL mapping and single nucleotide polymorphisms in FAE gene. Theor Appl Genet, 2004, 08: [2] Doyle J J, Doyle J L. Isolation of plant DNA from fresh tissue. Focus, 990, 2: 3 5

Microsoft Word - 17--刘秀秀_new_.doc

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