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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (11): 22-30    DOI: 10.13523/j.cb.20191103
研究报告     
异源表达CiRS基因拟南芥的黄酮代谢及抑菌能力研究 *
杨飞芸1,2,杨天瑞1,刘坤1,崔爽1,王瑞刚1,李国婧1,**()
1 内蒙古农业大学生命科学学院 内蒙古自治区植物逆境生理与分子生物学重点实验室 呼和浩特 010011
2 内蒙古农业大学食品科学与工程学院 呼和浩特 010018
Flavonoids Metabolism and Antimicrobial Activity of Arabidopsis Heterologous Expressing CiRS Gene
YANG Fei-yun1,2,YANG Tian-rui1,LIU Kun1,CUI Shuang1,WANG Rui-gang1,LI Guo-jing1,**()
1 College of Life Sciences, Inner Mongolia Agricultural University, Inner Mongolia Key Laboratory of Plant Stress Physiology and Molecular Biology, Hohhot 010011, China
2 College of Food Science and Engineering, Inner Mongolia Agricultural University, Hohhot 010018, China
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摘要：

白藜芦醇合成酶(resveratrol synthase, RS)是查耳酮合酶基因家族的一个重要酶,在植物体内催化白藜芦醇的生成。白藜芦醇是植物产生的一种非黄酮多酚类代谢产物,是植物在受到生物和非生物胁迫时产生的植物抗毒素,已证实具有多种生理活性。从转录组数据库中筛选获得注释为CHS基因的CDS序列,以中间锦鸡儿cDNA为模板,克隆得到基因全长。序列分析、系统进化分析和转该基因拟南芥研究结果表明,该基因为RS基因,因此将其命名为CiRS(GenBank登录号MF678590)。qRT-PCR检测分析发现,中间锦鸡儿CiRS基因的表达受到干旱、NaCl、紫外线等胁迫诱导。异源表达CiRS基因抑制了拟南芥自身AtCHS基因的表达。同时转CiRS基因拟南芥的抑菌活性强于野生型。这些结果均证实了中间锦鸡儿CiRS基因在转基因拟南芥中发挥了相应的功能。
关键词： 白藜芦醇合成酶;  中间锦鸡儿;  抑菌活性;  转基因拟南芥    
Abstract:

Resveratrol synthetase (RS), an important enzyme of chalcone synthase gene super family, catalyzes resveratrol production in plants. Resveratrol is a non-flavonoid polyphenol metabolite, which is a phytoalexin produced by plants under both biotic and abiotic stress. It has been proved that resveratrol has a variety of physiological activities.The CDS sequence annotated as CHS gene was obtained from the database of transcriptome, and the full length gene sequence was cloned using Caragana intermedia cDNA as template. Sequence, phylogenetic analysis and characteristic study of transgenic Arabidopsis indicated that the gene was a RS gene, then it was named CiRS. The expression of CiRS was induced by drought, NaCl and ultraviolet (UV) stresses as revealed by quantitative real-time PCR (qRT-PCR). Heterologous expression of CiRS gene inhibited the expression of endogenous AtCHS gene in transgenic Arabidopsis, meanwhile the antimicrobial activity of transgenic lines was stronger than that of wild type. All these results confirmed that the CiRS of C. intermedia played roles in transgenic Arabidopsis.
Key words: Resveratrol synthase    Caragana intermedia    Antimicrobial activity    Transgenic Arabidopsis
收稿日期: 2019-04-29 出版日期: 2019-12-17
ZTFLH:  Q786  
基金资助: * 内蒙古自治区自然科学基金(2017MS0354);内蒙古自治区科技创新引导项目(KCBJ2018012);内蒙古自治区科技计划(201802083)
通讯作者: 李国婧     E-mail: liguojing@imau.edu.cn
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引用本文:

杨飞芸,杨天瑞,刘坤,崔爽,王瑞刚,李国婧. 异源表达CiRS基因拟南芥的黄酮代谢及抑菌能力研究 *[J]. 中国生物工程杂志, 2019, 39(11): 22-30.

YANG Fei-yun,YANG Tian-rui,LIU Kun,CUI Shuang,WANG Rui-gang,LI Guo-jing. Flavonoids Metabolism and Antimicrobial Activity of Arabidopsis Heterologous Expressing CiRS Gene. China Biotechnology, 2019, 39(11): 22-30.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191103    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I11/22

图1  CiRS基因克隆电泳结果
图2  CiRS基因DNA全长及氨基酸序列
图3  CiRS与其他物种来源的RS和CHS构建的系统进化树
图4  不同胁迫处理下CiRS基因的表达分析
图5  转CiRS基因拟南芥AtCHS表达水平检测
图6  拟南芥叶片感染疫霉菌的实验结果
[1] 杨飞芸, 武燕燕, 崔爽 , 等. 异源表达CiRS基因通过生成白藜芦醇增强拟南芥的抗氧化能力. 中国生物工程杂志, 2017,37(12):27-33.
Yang F Y, Wu Y Y, Cui S , et al. Heterologous expression of CiRS gene enhances the antioxidant capacity of Arabidopsis by increasing the content of resveratrol. China Biotechnology, 2017,37(12):27-33.
[2] Jeandet P, Delaunois B, Conreux A , et al. Biosynjournal, metabolism, molecular engineering and biological functions of stilbene phytoalexins in plants. Biofactors, 2010,36(5):331-341.
doi: 10.1002/biof.108 pmid: 20726013
[3] Gambini J, Inglés M, Olaso G , et al. Properties of resveratrol: In vitro and in vivo studies about metabolism, bioavailability, and biological effects in animal models and humans. Oxidative Medicine and Cellular Longevity, 2015,2015:1-13.DOI: 10.1155/2015/837042.
doi: 10.1155/2015/837042 pmid: 29168580
[4] Basholli-Salihu M, Schuster R, Mulla D , et al. Bioconversion of piceid to resveratrol by selected probiotic cell extracts. Bioprocess Biosyst Eng, 2016,39(12):1879-1885.
doi: 10.1007/s00449-016-1662-1 pmid: 27497981
[5] Flieger J, Tatarczak-Michalewska M, Blicharska E . Characterization of the cis/trans isomerization of resveratrol by high-performance liquid chromatography. Analytical Letters, 2016,50(2):10.
doi: 10.1002/rcm.3316 pmid: 18023075
[6] Lu Y, Shao D Y, Shi J L , et al. Strategies for enhancing resveratrol production and the expression of pathway enzymes. Applied Microbiology & Biotechnology, 2016,100(17):7407-7421.
doi: 10.1111/gcb.14962 pmid: 31838767
[7] Shomura Y, Torayama I, Suh D Y , et al. Crystal structure of stilbene synthase from Arachis hypogaea. Proteins: Structure, Function, and Bioinformatics, 2005,60(4):803-806.
[8] Yu C K Y, Karin S, Jürgen S , et al. A stilbene synthase gene (SbSTS1) is involved in host and nonhost defense responses in sorghum. Plant Physiology, 2005,138(1):393-401.
doi: 10.1104/pp.105.059337 pmid: 15821144
[9] Hain R, Bieseler B, Kindl H , et al. Expression of a stilbene synthase gene in Nicotiana tabacum results in synjournal of the phytoalexin resveratrol. Plant Molecular Biology, 1990,15(2):325-335.
doi: 10.1007/bf00036918 pmid: 2103451
[10] Shi J L, He M Y, Cao J L , et al. The comparative analysis of the potential relationship between resveratrol and stilbene synthase gene family in the development stages of grapes (Vitis quinquangularis and Vitis vinifera). Plant Physiology & Biochemistry, 2014,74:24-32.
doi: 10.1016/j.plaphy.2019.12.002 pmid: 31837557
[11] Delaunois B, Cordelier S, Conreux A , et al. Molecular engineering of resveratrol in plants. Plant Biotechnology Journal, 2009,7(1):2-12.
doi: 10.1111/j.1467-7652.2008.00377.x pmid: 19021877
[12] Bai J, Kang T, Wu H D , et al. Relative contribution of photorespiration and antioxidative mechanisms in Caragana korshinskii under drought conditions across the Loess Plateau. Functional Plant Biology, 2017,44(11):1111-1123.
doi: 10.1071/FP17060
[13] Yang F Y, Yang T R, Liu K , et al. Analysis of metabolite accumulation related to pod color variation of Caragana intermedia. Molecules, 2019,24(4):717.
doi: 10.1038/sj.emboj.7600559 pmid: 15692567
[14] Luan G X, Wang H L, Lv H H , et al. Separation and purification of five flavone glucosides and one lignan from Caragana korshinskii Kom. by the combination of HSCCC and semi-preparative RPLC. Chromatographia, 2016,79(13):823-831.
doi: 10.1007/s10337-016-3090-4
[15] Zeng Z, Ji Z Y, Hu N , et al. A sensitive pre-column derivatization method for the analysis of free fatty acids by RP-HPLC with fluorescence detector and its application to Caragana species. Journal of Chromatography B, 2017,1064 : 151-159.
doi: 10.1016/j.jchromb.2017.07.037 pmid: 28927568
[16] 韩晓敏 . 中间锦鸡儿3个非生物胁迫相关转录因子的克隆与功能分析. 呼和浩特:内蒙古农业大学, 2015: 24-26.
Han X M . The cloning and analysis of three stress-related transcription factors from Caragana intermedia. Hohhot: Inner Mongolia Agricultural University, 2015: 24-26.
[17] 杨飞芸, 刘坤, 崔爽 , 等. 转CiCHS基因拟南芥的黄酮代谢及抗氧化能力分析. 西北植物学报, 2018,38(3):393-400.
Yang F Y, Liu K, Cui S , et al. Metabolism of flavonoids and the antioxidant capacity of transgenic Arabidopsis expressing CiCHS gene. Acta Botanica Boreali-Occidentalia Sinica, 2018,38(3):393-400.
[18] O’Dell M, Metzlaff M, Flavell R B . Post-transcriptional gene silencing of chalcone synthase in transgenic petunias, cytosine methylation and epigenetic variation. The Plant Journal, 1999,18(1):33-42.
doi: 10.1099/00221287-18-1-33 pmid: 13525625
[19] Shanker A K, Shanker C . Abiotic and biotic stress in plants: Recent advances and future perspectives. London: IntechOpen, 2016: 185-227.
doi: 10.1089/ars.2019.7819 pmid: 31701753
[20] Chong J L, Poutaraud A, Hugueney P . Metabolism and roles of stilbenes in plants. Plant Science, 2009,177(3):143-155.
doi: 10.1186/s12870-019-1935-3 pmid: 31387524
[21] Salehi B, Mishra A P, Nigam M , et al. Resveratrol: A double-edged sword in health benefits. Biomedicines , 2018,6(3):91.
doi: 10.3390/biomedicines6030091 pmid: 30205595
[22] Cho Y B, Jones S I, Vodkin L O . Mutations in Argonaute5 illuminate epistatic interactions of the K1 and I loci leading to saddle seed color patterns in Glycine max. The Plant Cell, 2017,29(4):708-725.
doi: 10.1105/tpc.17.00162 pmid: 28351993
[23] Espa?a L ,Heredia-Guerrero J A, Reina-Pinto J J, et al. Transient silencing of chalcone synthase during fruit ripening modifies tomato epidermal cells and cuticle properties. Plant Physiology, 2014,166(3):1371-1386.
doi: 10.1104/pp.114.246405
[24] Suzuki N, Rivero R M, Shulaev V , et al. Abiotic and biotic stress combinations. New Phytologist, 2014,203(1):32-43.
doi: 10.1111/nph.12797
[25] Tsuda K, Somssich I E . Transcriptional networks in plant immunity. New Phytologist, 2015,206(3):932-947.
doi: 10.1111/nph.13286 pmid: 25623163
[26] Dao T T H, Linthorst H J M, Verpoorte R . Chalcone synthase and its functions in plant resistance. Phytochemistry Reviews, 2011,10(3):397-412.
doi: 10.1007/s11101-011-9211-7
[27] Tang K, Zhan J C, Yang H R , et al. Changes of resveratrol and antioxidant enzymes during UV-induced plant defense response in peanut seedlings. Journal of Plant Physiology, 2010,167(2):95-102.
doi: 10.1016/j.jplph.2009.07.011 pmid: 19716623
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