SOD SNP % Cu /Zn-SOD. DAM- BE DNAStar % Mn /Fe-SOD. 6 1SNP /17. 9 bp
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1 http / /xuebao. jxau. edu. cn Acta Agriculturae Universitatis Jiangxiensis E - mail ndxb7775@ sina. com SOD SNP * GenBank Cu /Zn-SOD Mn /Fe-SOD 10 Cu /Zn-SOD Mn /Fe-SOD Cu /Zn-SOD 375 bp Mn /Fe-SOD 429 bp DAM- BE DNAStar Cu/Zn-SOD 74% 79% Mn /Fe-SOD 78% 81% Cu /Zn-SOD 21 SNPs 6 1SNP /17. 9 bp 5. 6% 4. 8% Mn /Fe-SOD 17 SNPs 3 1SNP /25. 2 bp 4. 0% 2. 1% Neighbour-Joining 10 Cu /Zn-SOD SOD SNP Q A Cloning of SOD Gene Segments and SNP Analysis in Evodia rutaecarpa WU Bo 1 GAO Dan 2 PAN Chao-mei 3 LUO Guang-ming 2 ZHANG Shou-wen 2* 1. Chinese Medicine Germplasm Resource Engineering Technology Research Center Jiangxi University of Traditional Chinese Medicine Nanchang China 2. College of Medicine Jiangxi University of Traditional Chinese Medicine Nanchang China 3. College of Pharmacy Guangzhou University of Chinese Medicine Guangzhou China Abstract Two pairs of degenerate primers were designed according to the two conserved regions on the a- mino acid sequences of Cu /Zn-SOD gene and Mn /Fe-SOD gene from kinds of plants reported in GenBank in order to clone the core fragments of these two genes in ten Evodia rutaecarpa s plants. The length of Cu /Zn- SOD gene and Mn /Fe-SOD gene were 375 bp and 429 bp respectively. Every nucleotide sequence of Cu /Zn- SOD gene and Mn / Fe-SOD gene encoded 125 amino acids and 143 amino acids respectively. The biology softwares of DAMBE and DNA Star5. 0 were used to analyze these sequences. The nucleotide similarity with other plants was above 74% and the amino acid similarity was above 79% at ten Cu /Zn-SOD gene fragments than other plants. The nucleotide similarity was above 78% and the amino acid similarity was above 81% in ten Mn / Fe-SOD gene fragments. Twenty-one sites of single nucleotides were discovered in the ten Cu / Zn-SOD BAI04B E - mail hero220@ 126. com *
2 6 SOD SNP 1207 gene fragments. Six amino acids sites transformed because of twenty-one single nucleotides variation. Seventeen variation sites of single nucleotides were discovered in the ten Mn / Fe-SOD gene fragments. Three amino acid sites transformed because of the variation of seventeen single nucleotides. Accoring to the calculation result the polymorphism frequences in Cu /Zn-SOD gene and Mn /Fe-SOD gene were 1SNP /17. 9 bp and 1SNP / bp the nucleotide degrees of variation were 5. 6% and 4. 0% the amino acid degrees of variation were 4. 8% and 2. 1% respectively. Genetic diversity of cloned ten Cu /Zn-SOD gene fragments was analyzed by using the clustering procedure of Neighbor-Joining. Key words Evodia rutaecarpa SOD gene cloning SNP Superoxide dismutase SOD EC Cu /Zn-SOD Mn /Fe-SOD SOD 2 Perl 3 Cu / Zn - SOD SOD SOD Cu /Zn-SOD Mn /Fe-SOD SOD 3 RAPD AFLP 7 SNP SNP 10 SOD GAP Escherichia coli JM10 peasy-t1 Taq PCR Promge dntps DNA marker RNA RNA 8 OD 260 /OD 280 OD 260 /OD GenBank Cu /Zn-SOD 1 WCZ1 5 - GGWCCNACMACTGTDACTGGA - 3 WCZ2 5 - AARTCCAAYAARACCACAAGC - 3 GenBank Mn /Fe-SOD WMF1 5 - CAGAARCAYCAYCARACNTAYGT -3 WMF2 5
3 Tab. 1 The name of test materials and their sources No. Test cultivars Name Source 1 Evodia rutaecarpa Juss. Benth. 2 E. rutaecarpa Juss. Benth. var. officinalis Dode Huang 3 E. rutaecarpa Juss. Benth. var. officinalis Dode Huang 4 E. rutaecarpa Juss. Benth. var. bodinieri Dode Huang 5 E. rutaecarpa Juss. Benth. var. bodinieri Dode Huang 6 E. rutaecarpa Juss. Benth. var. officinalis Dode Huang 7 E. rutaecarpa Juss. Benth. var. officinalis Dode Huang 8 E. rutaecarpa Juss. Benth. var. officinalis Dode Huang 9 E. rutaecarpa Juss. Benth. var. officinalis Dode Huang 10 Evodia meliifolia Benth - RTARTANGCRTGYTCCCA - 3 Invitrogen RNA cdna Invitrogene M-MLV cdna Cu /Zn-SOD Mn /Fe-SOD PCR 50 μl 10 Reaction buffer 5 μl Mg μl dntps 2. 5 mmol /L 4. 5 μl 10 μmol /L 1 μl 10 μmol /L 1 μl cdna7. 5 μl Taq DNA polymerase 5 U /μl μl ddh 2 O μl Cu /Zn - SOD PCR 94 5 min 94 1 min 45 1 min 72 1 min min 4 Mn /Fe-SOD PCR 94 5 min 94 1 min 50 1 min 72 1 min min 4 PCR 12 g /L peasy-t1 JM109 LB EcoRⅠ HindⅢ DAMBE DNAStar Cu /Zn-SOD PCR 375 bp EcoRⅠ HindⅢ 375 bp Fig. 1 Cu /Zn-SOD PCR Marker bp Marker PCR product of Cu /Zn-SOD gene fragments from Evodia rutaecarpa 2 Cu /Zn-SOD Marker bp Marker Fig. 2 Detection by EcoRⅠand HindⅢ digestion for recombinant plasmid from Evodia rutaecarpa Cu / Zn-SOD gene fragments
4 6 SOD SNP Fig. 3 Mn /Fe-SOD PCR Marker bp Marker PCR product of Mn /Fe-SOD gene fragments from Evodia rutaecarpa 4 Mn /Fe-SOD Marker bp Marker Fig. 4 Detection by EcoRⅠand Hind Ⅲ digestion for recombinant plasmid from Evodia rutaecarpa Mn / Fe-SOD gene fragments Mn /Fe-SOD PCR 430 bp EcoRⅠ HindⅢ 429 bp Cu /Zn-SOD 10 Cu /Zn- SOD 375 bp 125 GenBank JN JN GenBank Cu /Zn-SOD 74% 79% 92% Mn /Fe-SOD 10 Mn /Fe- SOD 429 bp 143 GenBank JN JN GenBank Mn /Fe-SOD 78% 81% 93% 2. 3 SOD SNP Cu /Zn-SOD SNP DNAStar5. 0 SNP bp SNP / bp Tab. 2 2 Cu /Zn-SOD Variation sites of single nucleotides in the fragments of Cu / Zn-SOD gene No A T T T A A A C C A T G C A G C A C C T T 2 A T C C C A A C C A T T C A A C G T C C A 3 A T C T C A A C C A A G C A G C A T C C C 4 A T C T C A A C C A A T C A A C A C C C C 5 A A C C C A A C C A A T C A A C G C C C C 6 A A C C C T A C C A T G T A A C A C C C C 7 A A C C C T A C C G T T T A A C A T C C C 8 A A C C C T A C C G T T T A A C A C C C C 9 A A C C C T A C C A T G T A A C A C C C C 10 C A T T G A T T T A A T T C A G A T T C T
5 Tab. 3 3 Cu /Zn-SOD Variation sites of amino acids in the fragments of Cu /Zn-SOD gene No L E N G T I 2 P D N S T V 3 L E N G T I 4 L D N S T I 5 P D N S T V 6 P E N S T I 7 P D N S T I 8 P D N S T I 9 P E N S T I 10 L D T S S I Tab. 4 4 Mn /Fe-SOD Variation sites of single nucleotides in the fragments of Mn / Fe-SOD gene No A C C T C T T A C G T A A G G C T 2 G C C C C T T G T G T G G G A C C 3 G C C C C T T G T G T G A G A C C 4 G C C T C T T G C T T A A G G C T 5 G C C T T T T G C T T A A G G C T 6 G T T T T T T G T G T G G G A C C 7 A T T T T T T G T T T G G G A C C 8 G T T T T T T G C G T G G G A C T 9 G T T T C T T G T G T G G G A C T 10 G T T C T G A G C T C G G T A A T Mn /Fe-SOD SNP DNAStar5. 0 SNP bp SNP / bp SOD 5 Mn /Fe-SOD DAMBE Tab. 5 Variation sites of amino acids in the fragments of Mn /Fe-SOD gene Cu /Zn-SOD No F I N Neighbour-Joining 2 F V S 3 F V S 4 F V N 5 F V N 6 F V S 7 F V S 8 F V S 3 9 F V S 1 SOD 10 D V S Cu / Zn-SOD Mn-SOD Fe-SOD 3 Mn-SOD Fe-SOD Cu /Zn-SOD
6 6 SOD SNP Cu / Zn-SOD Mn /Fe-SOD 5. 9% 5. 6% 2 SOD SNP 10 Cu /Zn-SOD Mn /Fe-SOD SNP Cu /Zn-SOD 375 bp 21 6 Mn /Fe-SOD 429 bp 17 3 SOD 11 6 Mn /Fe - SOD Cu /Zn-SOD Cu /Zn-SOD 13 Cu /Zn-SOD Cu /Zn-SOD Cu /Zn-SOD SNP 10 Cu /Zn-SOD 10 RAPD AFLP SNP Kota 14 7 SNP TaFLD 16 Mi-1 SNP 438 F 2 SNP 17 SNP 1 Bubliy O A Loechcke V. Correlated responses to selection for stress resistance and longevity in a laboratory population of Drosophila melangoaster J. Journal of Evolutionary Biology Tsang E W Bowler C Herouart D et al. Differential regulation of superoxide dismutases in plants exposed to environmental stress J. Plant Cell Perl T R Galun E. The tomato Cu Zn superoxide dismutase genes are developmnentally regulated and respond to light and stress J. Plant Mol Biol Priya P Venkatachalam P Thulaseedharan A. Molecular cloning and characterization of the rubber elongation factor gene and its promoter sequence from rubber tree Hevea brasiliensis A gene involved in rubber biosynthesis J. Plant Science Andreas E Voloudakis Philippe Marmey Etienne Delannoy et al. Molecular cloning and characterization of Gosspium hirsutum superoxide dismutase genes during cotton-xanthomonas campestris pv malvacearum interaction J. Physiological and Molecular Plant Pathology Cu /Zn MrSOD1 cdna J AFLP J DNA RNA J SOD J SOD J SOD SNP J J Cu /Zn-SOD J Kota R Varsheny R K Thiel T et al. Generation and comparison of EST-derived SSRs and SNPs in barley Hordemu uulgare L. J. Hereditas TaFLD J Mi-1 SNP J RAPD J
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