stage during RNAi treatment These results indicated that the peroxidase gene Rsprx1 might be involved in anthocyanin synthetic metabolism Key w

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1 ISSN CN / Q http/ /cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 872 RNAi Rsprx1 * ( 河南师范大学生命科学学院, 河南新乡 ) 过氧化物酶是一类广泛存在于生物中的氧化还原酶, 被认为参与植物花青素的代谢本实验利用 RNAi 技术, 干扰萝卜过氧化物酶基因 Rsprx1 表达结果表明,RNAi 干扰载体的萝卜植株中过氧化物酶基因 ( Rsprx1) 表达被抑制, 过氧化物酶活性显著降低, 过氧化物酶同工酶条带减少 ; 而花青素含量在处理第 9 d 达到最大值 ; 花青苷种类和含量有较大变化 : 天竺葵素 -3- 阿魏酰葡糖苷 - 5- 丙二酰基葡糖苷天竺葵素 -3-ρ- 香豆酰二葡糖苷 -5- 丙二酰基葡糖苷天竺葵素 -3- 阿魏酰二葡糖苷 -5- 丙二酰基葡糖苷和天竺葵素 -3- 二 ρ- 香豆酰二葡糖苷 -5- 丙二酰基葡糖苷含量升高 ; 天竺葵素 - 3- 二葡糖苷 -5- 葡糖苷天竺葵素 -3- 葡糖苷 -5- 葡糖苷和天竺葵素 -3- 酰化二葡糖苷 -5- 葡糖苷含量降低 ; 花青素合成相关基因 ( Chs Chi Dfr F3h 和 Ldox) 及转录因子 ( Tt8) 的 mrna 表达水平在 RNAi 处理后早期有明显上调这些结果均表明, 萝卜过氧化物酶 Rsprx1 参与花青素的合成代谢过氧化物酶 ; 花青素 ; RNA 干扰 ; 萝卜 ; 次生代谢 Q RNA Interference of Peroxidase Gene Rsprx1 Increased Anthocyanidin Accumulation in Chinese Red Radish MU ChunWANG LiJIA Xiao-LinWANG Ke-KePENG Fang-LinGUO Li-JieWANG Lin-Song * College of Life SciencesHenan Normal UniversityXinxiang HenanChina Abstract Peroxidase POD is an oxidoreductase that participates the process of anthocyanidin metabolism We knocked down the expression of peroxidase gene Rsprx1 in Chinese red radish Raphanus sativus L by RNA interference RNAi The peroxidase activity was significantly reduced in the RNAi group than that in the controls as shown by the weaker POD isoenzyme bands The anthocyanin content increased and reached the maximum on day 9 in the Rsprx1 knockdown group The types of anthocyanin were also changed The contents of pelargonidin 3-feruloyl glucoside-5-malonlyl glucosidepelargonidin 3-ρ-coumaroyl diglucoside-5-malonoyl glucosidepelargonidin3-feruloyl diglucoside-5-malonoyl glucoside and pelargonidin 3-ρ-coumaroyl ρ-coumaroyl diglucoside-5-malonoyl glucoside were increasedand the content of pelargonidin 3-diglucoside-5-glucoside pelargonidin 3- glucoside-5-glucoside and pelargonidin 3-acyl diglucoside-5- glucoside were decreased as detected using high-performance liquid chromatography The mrna levels of anthocyanin synthesis genes such as chalcone synthasechalcone isomerasedihydroflavonol 4- reductaseflavanone 3-hydroxylase leucoanthocyanidin dioxygenase and transcription factors Tt8were significantly increased at the early No No No 12A * Tel wls@ htu cn ReceivedFebruary AcceptedJune Supported by National Natural Science Foundation of China No Key Discipline of Biochemistry and Molecular Biology of Henan Province No Research Fund of the Major Program of Science and Technology in Henan Provincial Committee of EducationNo 12A * Corresponding author Tel wls@ htu cn

2 stage during RNAi treatment These results indicated that the peroxidase gene Rsprx1 might be involved in anthocyanin synthetic metabolism Key words peroxidaseanthocyanidinrna interference RNAi Raphanus sativus L secondary metabolism anthocyanidin anthocyanin part27- RsPrx1i 12 Rsprx1 1 Raphanus sativus L 2 phenylalanine ammonialyase PAL 3 d 30 d 1 2 POD 3 chalcone synthase CHS ph mol /L K 2 HPO 4 -NaH 2 PO 4 chalcone isomerase CHI r /min 4 20 dihydroflavonol 4-reductase DFR 3-min - 20 POD flavanone 3-hydroxylase F3H U /g FWfresh weight leucoanthocyanidin dioxygenase LDOX -8 transparent 1 3 POD testa8 genett8 Myb myeloblastosis 16 - transcription factors Myb 7 % ~ 12 % peroxidase POD h RNA POD r /min 4 C 10 min520 8 POD nm 18 A 520 /g FW POD POD 10 9 d POD POD g 2 ml 5 % POD Rsprx1 12 h r /min 15 min 11 POD Park 19 part27-rsprx1i 12 C cm 250 cm 40 5 % RNAi POD 95 5 V /V 5 % 95 5 V /V part g POD 0 3 g 1 8 ml 1 % 20 μl 520 nm 1 6 POD RT-PCR TRIZOL R sativus L RNA 2 μg RNA Access RT-PCR 2010 ~ 2011 PCR 8 Actin d Table 1 PCR 13 Hoffmann s 50 ~ s 72 1 min 30 PCR 1 %

3 9 RNAi Rsprx1 869 Table 1 Oligonucleotide primers used for semi-quantitative RT-PCR analysis Genes Forward primer 5' 3' Reverse primer 5' 3' Actin8 TCGTTGGTCGTCCTCGAC GGGTTGAGAGGAGCCTCA Rsprx1 GGATCCATGGGGTTTTCTCCTTCGTTT GAGCTCTCAGATAGAACTCACAACACCATC Rsprxn CGB CTT CAY TTY CAT GACTG GCT YTT CCA AAT GTG TGA CC Pal CATCATCCTGGGCAAATTGAAGC TGTTGCTCGGCGCTCTGGACATG Chs GTTCAAGCGCATGTGCGACAAGTCG TCTCTCCGACAGATGTGTCAGG Chi GTTCAAGCGCATGTGCGACAAGTCG TCTCTCCGACAGATGTGTCAGG F3h ATATTTCAAGTTGTAGATCATGGAGTG TGGTCGGCGTTTTTAAAGCTGCC Dfr ATGGTTAGTCAGAAAGAGACCG GTCTTATGATCGAGTAATGCGC Ldox AGACCATGGTTGCGGTTGAAAGAGTTG ATCATTTTTCTCGGATACCAATTCCTC Tt8 ATGGATGAATCAAGTATTATTCCGG CTCCACGTGGCAAACGATGATTGG Myb GAGGAAAGGTGCATGGAC CCTAGAAGCTTATGAAGCC 1 7 SPSS 13 0 x 珋 ± s ANVOA LSD Rsprx1 POD Rsprx1 POD Fig 1 Effect of suppressing Rsprx1 gene on activities of RNAi POD in Chinese red radish Samples were harvested Fig 1 POD every 3 days after radish fleshy root were injected by water 6 d POD empty vector part-27and RNAi vector part-rsprx1i respectively Two vectors were used in Agrobacterium P < d suspension POD activity was determined at 470 nm using P < d guaiacol as substrates expressed as 10 3 U /g FW Values are P < 0 01 POD expressed as means ± SD of three determinations from 15 d ~ 27 d separate experiments * P < 0 05 ** P < 0 01compared with P < 0 05 POD Fig 2 A B C part-27 group DPOD POD Rsprx1 9 d Fig 2 2 B 9 d C POD d POD POD Rsprx Rsprx RNAi Fig 3-3-ρ d 9 d P < ρ d Fig 4

870 29 Fig 2 Effect of suppressing Rsprx1 gene on the isozymes of POD in Chinese red radish Samples were extracted from the Chinese red radish fleshy root on day 18212427 after treatment and

4 Fig 2 Effect of suppressing Rsprx1 gene on the isozymes of POD in Chinese red radish Samples were extracted from the Chinese red radish fleshy root on day after treatment and separated by 7 % -12 % non-denaturing gradient-sds-page 14 POD samples were stained using biphenyl amine /H 2 O 2 Arrowsbrackets and numbers indicate different POD isozymes Wwater groupp27part-27 group prpart-rsprx1i group Fig 3 Effect of suppressing Rsprx1 gene on the content of Anthocyanidin in Chinese red radish Samples were harvested every 3 days after radish fleshy root were injected by waterempty vector part-27 and RNAi vector partrsprx1i respectively Two vectors were used in Agrobacterium suspension Samples were extracted with 1 % HCl ethanol and measured at 520 nm using spectrophotometerexpressed as A 520 /g FW Values are expressed as means ± SD of three determinations from separate experiments * P < 0 05 ** P < 0 01compared with part-27 group Fig 4 The anthocyanidin derivatives of red radish fleshy root on day 9 after treatment Samples were harvested on day 9 after treatment AHPLC profiles of the anthocyanidin in Chinese red radish 1 B The proportion of the anthocyanidin components based on HPLC peak area data The bar graph indicated the percentage of main anthocyanidins among the three groups 1pelargonidin 3-diglucoside-5-glucoside 2 pelargonidin 3-glucoside-5- glucoside10pelargonidin 3-acyl diglucoside-5-glucosidec 14pelargonidin 3-feruloyl glucoside-5-malonly glucoside 21 pelargonidin 3-ρ-coumaroyl diglucoside-5- malonoyl glucoside 22 pelargonidin 3-feruloyl diglucoside-5- malonoyl glucoside28 pelargonidin 3-ρ-coumaroyl ρ- coumaroyl diglucoside-5-malonoyl glucoside W water groupp27part-27 groupprpart-rsprx1i group POD 2 3 Rsprx1 9 d Chs Chi Dfr Ldox Tt8 9 d 21 d POD 21 d 9 d 21 d Fig 5 RT-PCR Rsprx1 9 d 21 d 3 2 POD Rsprxn Rsprx1 20

9 RNAi Rsprx1 871 Fig 5 RT-PCR analysis of expression of Rsprx1Rsprxn and anthocyanidin biosynthesis genes in Chinses red radish Samples were harvest on day 9 and 21 after treatment Total RNA was

5 9 RNAi Rsprx1 871 Fig 5 RT-PCR analysis of expression of Rsprx1Rsprxn and anthocyanidin biosynthesis genes in Chinses red radish Samples were harvest on day 9 and 21 after treatment Total RNA was isolated from radish fleshy root using Trizol reagent protocol The cdna was synthesized using the oligodtprimer Oligonucleotide primers used for semiquantitative RT-PCR analysis were listed in Table 1 Relative transcription levels of genes were quantified with a PhosphorImager normalized against Actin8 Values are the means ± SD of three determinations from separate experiments Wwater groupp27part-27 groupprpart-rsprx1i group POD Rsprxn 21 POD POD Rsprx1 Rsprxn 8 POD POD Rsprx1 Rsprx1 6 POD 24 RNAi POD Rsprx1 Rsprx1 POD Rsprx1 26 POD 9 Fig Rsprx1 POD POD Tt8 Rsprx1 Chs Chi Dfr Ldox POD Rsprx1 9 POD Fig 5 5 RT-PCR References POD 1 Wu XPrior R L Identification and characterization of

6 Anthocyanins by high-performance liquid chromatographyelectrospray ionization-tandem mass spectrometry in common foods in the United Statesvegetablesnutsand grainsj J Agric Food Chem Matsufuji H Otsuki T Takeda Tet al Identification of reaction products of acylated anthocyanins from red radish with peroxyl radicals J J Agric Food Chem Cooper-Driver G A Contributions of Jeffrey Harborne and coworkers to the study of anthocyaninsj Phytochemistry Cominelli EGusmaroli GAllegra Det al Expression analysis of anthocyanin regulatory genes in response to different light qualities in Arabidopsis thaliana J J Plant Physiol Yuan YChiu L WLi L Transcriptional regulation of anthocyanin biosynthesis in red cabbagej Planta Zhang Z Pang X Ddan Xet al Role of peroxidase in anthocyanin degradation in litchi fruit pericarp J Food Chem Oren-Shamir M Does anthocyanin degradation play a significant role in determining pigment concentration in plants J Plant Sci Vaknin HBar-Akiva AOvadia Ret al Active anthocyanin degradation in Brunfelsia calycina yesterday- today-tomorrow flowers J Planta Wang LBurhennea KKristensena B Ket al Purification and cloning of a Chinese red radish peroxidase that metabolise pelargonidin and forms a gene family in BrassicaceaeJ Gene Nquyen Q TMerlo M EMedema M Het al Metabolomics J methods for the synthetic biology of secondary metabolismj Wang LWang L SSun X Det al Isoenzymic and histochemical analysis of peroxidase in root of Chinese red radish during vegetative growth J Chin J Biochem Mol Biol RsPrx1 24Passardi FCosio CPenel Cet al Peroxidases have more J functions than a Swiss army knife J Plant Cell Rep Wang Y FWang LPeng G Set al Enhancement of oxidative stress tolerance in Arabidopsis thaliana by gene transfer of radish peroxidasej Chin J Biochem Mol Biol RNA J Yang S HWang LWang Y Fet al Construct for radish peroxidase RNA interference vector and identification J Acta Agric Boreali-occid Sin Spolaore STrainotti LCasadoro G A simple protocol for transient gene expression in ripe fleshy fruit mediated by Agrobacterium J J Exp Bot Hoffmann TKalinowski GSchwab W RNAi-induced silencing of gene expression in strawberry fruit Fragaria ananassaby agroinfiltrationa rapid assay for gene function analysisj Plant J Lee M YKim S S Characteristics of six isoperoxidases from Korean radish root J Phytochemistry PAGE J Wan Y LYang X P Cao B Set al Purity identification of watermelon hybrid by gradient polyacrylamide gel electrophoresisj Jiangsu J Agric Sci M Hu Neng-ShuWan Xian-Guo Technology and Application for Isoenzyme M ChangshaHunan Scientific Technology Press Wang L SSun X DCao Yet al Antioxidant and pro-oxidant properties of acylated pelargonidin derivatives extracted from red radish Raphanus sativus var nigerbrassicaceae J Food Chem Toxicol Park N IXu HLi X Het al Anthocyanin accumulation and expression of anthocyanin biosynthetic genes in radish Raphanus sativus J J Agric Food Chem J Yan X FWang YLi Y M Plant secondary metabolism and its response to environment J Acta Ecol Sin J Chen X Y Plant secondary metabolismj World Sci Technol Res Dev FEBS Lett Yang C QFang XWu X M et al Transcriptional regulation of plant secondary metabolismj J Integr Plant Biol cdna J Wang L SLi X JLi F Jet al cdna partial cloning of peroxidase genes from Raphanus sativus L and their expression in floral stage J Chin Veg Matsufuji HKido HMisawa Het al Stability to lightheat and hydrogen peroxide at different ph values and DPPH radical scavenging activity of acylated anthocyanins from red radish extract J J Agric Food Chem

380 研究论文发酵天数双乙酰测定 : 参照 GB4927-4928-2001 标准发酵液中的化学成分的测定 : 采用 GC-8A 型气相色谱测定 1.5 离子注入方法 [6] 把待处理的菌株细胞均匀涂

380 研究论文发酵天数双乙酰测定 : 参照 GB4927-4928-2001 标准发酵液中的化学成分的测定 : 采用 GC-8A 型气相色谱测定 1.5 离子注入方法 [6] 把待处理的菌株细胞均匀涂中国细胞生物学学报 Chinese Journal of Cell Biology 2011, 33(4): 379-384 http://www.cjcb.org 全小麦啤酒酵母菌株的诱变育种及应用研究袁仲 1* 张百胜 1 张慎举 1 马绮云 2 陈柯羽 2 ( 1 商丘职业技术学院园林食品加工系,