应用抑制性差减杂交技术筛选锦橙CTV应答基因

doi:10.3864/j.issn.0578-1752.2013.16.012

中国农业科学 ›› 2013, Vol. 46 ›› Issue (16): 3413-3423.doi: 10.3864/j.issn.0578-1752.2013.16.012

应用抑制性差减杂交技术筛选锦橙CTV应答基因

程春振12, 曾继吾2, 贝学军12, 吴波12, 阳佳位3, 张永艳12, 姜波2, 朱世平1, 闫树堂1, 钟云2, 钟广炎2

1.西南大学园艺园林学院/柑橘研究所，重庆 400715
2.广东省农业科学院果树研究所，广州 510640
3.四川省荣县经济作物站，四川自贡 643100

收稿日期:2013-03-25 出版日期:2013-08-15 发布日期:2013-05-27
通讯作者: 通信作者钟云，E-mail：zhongyun99cn@163.com。通信作者钟广炎，E-mail：gy_zhong@163.com
作者简介:程春振，Tel：020-38765854；E-mail：ld0532cheng@126.com
基金资助:
科技部国际合作项目（2012DF30610）、农业部公益性行业（农业）科研专项(201203075-07)、“十二五”国家科技计划课题（2011AA100205）、广东省科技计划项目（2012A020200016，2012B091100169）

Screening of ‘Jincheng’ Orange CTV Response Genes Using Subtractive Suppression Hybridization

CHENG Chun-Zhen-12, ZENG Ji-Wu-2, BEI Xue-Jun-12, WU Bo-12, YANG Jia-Wei-3, ZHANG Yong-Yan-12, JIANG Bo-2, ZHU Shi-Ping-1, YAN Shu-Tang-1, ZHONG Yun-2, ZHONG Guang-Yan-2

1.College of Horticulture and Landscape Architecture/Citrus Research Institute, Southwest University, Chongqing 400715
2.Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences, Guangzhou 510640
3.Economic Crops Station, Agricultural Bureau of Rongxian, Zigong 643100, Sichuan

Received:2013-03-25 Online:2013-08-15 Published:2013-05-27

摘要/Abstract

摘要： 【目的】探讨易感柑橘衰退病病毒（Citrus tristeza virus，CTV）品种锦橙CTV应答分子机理，筛选和分析CTV应答基因。【方法】以嫁接CTV强毒株TRL514的锦橙阳性材料叶片为试验方（Tester），用嫁接无毒枝条的CTV阴性锦橙叶片为驱动方（Driver），构建CTV侵染的锦橙的正向差减文库。【结果】随机挑选850个阳性克隆测序，其中742条测序成功，去除载体片段和接头序列后得到有效EST 692条。将所有EST序列对应到柑橘的预测基因组转录物，共涉及137个柑橘基因。应用BLAST2GO对这些特异性表达的基因进行功能归类和KEGG分析。结果表明，受CTV侵染的影响，锦橙能量代谢相关基因上调明显，与光合器官碳固定、氮代谢、淀粉和蔗糖代谢等途径相关的EST出现频率较高。另外，与抗逆防御以及苯基丙氨酸合成、类苯基丙烷代谢、类胡萝卜素合成等与植物防御素类物质合成密切相关的代谢途径相关基因较多。【结论】由于CTV的侵染，CTV易感品种锦橙基础代谢改变明显，须通过增强能量代谢和提高自身防御能力来维持自身生长和发育。

关键词: 柑橘 , 抑制性差减杂交技术 , 差异表达基因 , CTV

Abstract: 【Objective】The objective of this study is to make clear the molecular mechanism and screen citrus tristeza virus (CTV) response genes in CTV susceptive citrus species ‘Jincheng’ orange [Citrus sinensis (L) Osbeck].【Method】 A suppression subtractive hybridization (SSH) library of ‘Jincheng’ orange was successfully constructed by using the virus-free leaves as Driver and those infected with CTV as Tester. 【Result】After sequencing of the randomly picked 850 clones, 742 clones were successfully sequenced and a total of 692 ESTs were obtained after Vectorscreen and adaptors deletion. All these ESTs were then matched to citrus genome transcripts and a total of 137 genes were identified. BLAST2GO gene ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis results showed that the basic energy metabolisms of ‘Jincheng’ orange were significantly influenced with differentially expressed genes (DEGs) involving in carbon fixation in photosynthetic organisms, nitrogen metabolism, starch and sucrose metabolism, etc. Moreover, DEGs largely involved in defense and resistance and some plant defensins synthesis related pathways such as carotenoid biosynthesis, phenylpropanoid biosynthesis, and phenylalanine metabolism. 【Conclusion】Results generated in this study suggested that CTV susceptible species ‘Jincheng’ orange reacted severely to CTV infection to maintain its growth and development by elevating energy metabolisms and enhancing the expression of stress resistance related genes.

Key words: citrus , suppression subtractive hybridization (SSH) , differentially expressed genes (DEGs) , CTV

程春振12, 曾继吾2, 贝学军12, 吴波12, 阳佳位3, 张永艳12, 姜波2, 朱世平1, 闫树堂1, 钟云2, 钟广炎2. 应用抑制性差减杂交技术筛选锦橙CTV应答基因[J]. 中国农业科学, 2013, 46(16): 3413-3423.

CHENG Chun-Zhen-12, ZENG Ji-Wu-2, BEI Xue-Jun-12, WU Bo-12, YANG Jia-Wei-3, ZHANG Yong-Yan-12, JIANG Bo-2, ZHU Shi-Ping-1, YAN Shu-Tang-1, ZHONG Yun-2, ZHONG Guang-Yan-2. Screening of ‘Jincheng’ Orange CTV Response Genes Using Subtractive Suppression Hybridization[J]. Scientia Agricultura Sinica, 2013, 46(16): 3413-3423.

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参考文献

[1]Zhou Y, Zhou C, Song Z, Liu K, Yang F. Characterization of citrus tristeza virus isolates by indicators and molecular biology methods. Agricultural Sciences in China, 2007, 6: 573-579.

[2]Zhou C Y, Hailstones D L, Broadbent P, Connor R, Bowyer J. Studies on mild strain cross protection against stem-pitting citrus tristeza virus. Fifteenth IOCV Conference, Citrus Tristeza Virus, 2002: 151-157.

[3]Diatchenko L, Lau Y F, Campbell A P, Chenchik A, Moqadam F, Huang B, Lukyanov S, Gurskaya N, Sverdlov E D, Siebert P D. Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proceedings of the National Academy of Sciences of the United States of America, 1996, 93(12): 6025-6030.

[4]Randall P J, Bouma D. Zinc deficiency, carbonic anhydrase, and photosynthesis in leaves of spinach. Plant Physiology, 1973, 52: 229-232.

[5]Karlsson J, Clarke a K, Chen Z Y, Hugghins S Y, Park Y I, Husic H D, MoroneyJ V, Samuelsson G. A novel alpha-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2. The EMBO Journal, 1998, 17: 1208-1216.

[6]Gandía M, Conesa A, Ancillo G, Gadea J, Forment J, Pallás V, Flores R, Duran-Vila N, Moren P, Guerri J. Transcriptional response of Citrus aurantifolia to infection by citrus tristeza virus. Virology, 2007, 367: 298-306.

[7]Samanani N, Facchini P J. Purification and characterization of norcoclaurine synthase. The first committed enzyme in benzylisoquinoline alkaloid biosynthesis in plants. The Journal of Biological Chemistry, 2002, 277: 33878-33883.

[8]Samanani N, Liscombe D K, Facchini P J. Molecular cloning and characterization of norcoclaurine synthase, an enzyme catalyzing the first committed step in benzylisoquinoline alkaloid biosynthesis. The Plant Journal, 2004, 40: 302-313.

[9]Lee E J, Facchini P. Norcoclaurine synthase is a member of the pathogenesis-related 10/Bet v1 protein family. The Plant Cell, 2010, 22: 3489-3503.

[10]Bonas U, Lahaye T. Plant disease resistance triggered by pathogen- derived molecules: refined models of specific recognition. Current Opinion in Microbiology, 2002, 5: 44-50.

[11]Aarts N, Metz M, Holub E, Staskawicz B J, Daniels M J, Parker J E. Different requirements for EDS1 and NDR1 by disease resistance genes define at least two R gene-mediated signaling pathways in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95: 10306-10311.

[12]De Hoff P L, Brill L M, Hirsch A M. Plant lectins: the ties that bind in root symbiosis and plant defense. Molecular Genetics and Genomics, 2009, 282: 1-15.

[13]Peumans W J, Van Damme E J. Lectins as plant defense proteins. Plant Physiology, 1995, 109: 347-352.

[14]Dowd C, Wilson I.W, McFadden H. Gene expression profile changes in cotton root and hypocotyl tissues in response to infection with Fusarium oxysporum f. sp. vasinfectum. Molecular Plant-Microbe Interactions, 2004, 17: 654-667.

[15]Sun H, Kim M K, Pulla R K, Kim Y J, Yang D C. Isolation and expression analysis of a novel major latex-like protein (MLP151) gene from Panax ginseng. Molecular Biology Reports, 2010, 37: 2215-2222.

[16]Lima L S, Gramacho K P, Carels N, Novais R, Gaiotto F A, Lopes U V, Gesteira A S, Zaidan H A, Cascardo J C M, Pires J L, Micheli F. Single nucleotide polymorphisms from Theobroma cacao expressed sequence tags associated with ‘witches’ broom disease in cacao. Genetics and Molecular Research, 2009, 8: 799-808.

[17]Harada E, Kim J-A, Meyer A J, Hell R, Clemens S, Choi Y E. Expression pro?ling of tobacco leaf trichomes identi?es genes for biotic and abiotic stresses. Plant Cell Physiology, 2010, 51(10): 1627-1637.

[18]Fan J, Gao X, Yang Y W, Deng W, Li Z G. Molecular cloning and characterization of a NAC-like gene in “Navel” orange fruit response to postharvest stresses. Plant Molecular Biology Reporter, 2007, 25: 145-153.

[19]Zhong R, Richardson E A, Ye Z H. Two NAC domain transcription factors, SND1 and NST1, function redundantly in regulation of secondary wall synthesis in fibers of Arabidopsis. Planta, 2007, 225: 1603-1611.

[20]Yoo S Y, Kim Y, Kim S Y, Lee J S, Ahn J H. Control of flowering time and cold response by a NAC-domain protein in Arabidopsis. PLOS one, 2007, 2: e642.

[21]Uauy C, Distelfeld A, Fahima T, Blechl A, Dubcovsky J. A NAC gene regulating senescence improves grain protein, zinc, and iron content in wheat. Science, 2006, 314: 1298-1301.

[22]Selth L A, Dogra SC, Rasheed M S, Healy H, Randles J W, Rezaian M A. A NAC domain protein interacts with tomato leaf curl virus replication accessory protein and enhances viral replication. The Plant Cell, 2005, 17: 311-325.

[23]Wu K, Rooney M F, Ferl R J. The Arabidopsis 14-3-3 multigene family. Plant Physiology, 1997, 114: 1421-1431.

[24]Delille J M, Sehnke P C, Ferl R J. The Arabidopsis 14-3-3 family of signaling regulators. Plant Physiology, 2001, 126: 35-38.

[25]Roberts M R, Bowles D J. Fusicoccin, 14-3-3 proteins, and defense responses in tomato plants. Plant Physiology, 1999, 119: 1243-1250.

[26]Lapointe G, Luckevich M D, Cloutier M, Séguin A. 14-3-3 gene family in hybrid poplar and its involvement in tree defence against athogens. Journal of Experimental Botany, 2001, 52: 1331-1338.

[27]Koo A J K, Cooke T F, Howe G. Cytochrome P450 CYP94B3 mediates catabolism and inactivation of the plant hormone jasmonoyl-L-isoleucine. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108: 9298-9303.

[28]Rupasinghe S, Schuler M. Homology modeling of plant cytochrome P450s. Phytochemistry Reviews, 2006, 5: 473-505.

[29]Schuler M, Duan H, Bilgin M, Ali S. Arabidopsis cytochrome P450s through the looking glass: a window on plant biochemistry. Phytochemistry Reviews, 2006, 5: 205-237.

[30]Saito S, Hirai N, Matsumoto C, Ohigashi H, Ohta D. Arabidopsis CYP707As encode (+)-abscisic acid 8’-hydroxylase , a key enzyme in the oxidative catabolism of abscisic acid. Plant Physiology, 2004, 134: 1439-1449.

[31]Krochko J E, Abrams G D, Loewen M K, Abrams S R, Cutler A. (+) -Abscisic acid 8’ -hydroxylase is a cytochrome. Plant Physiology, 1998, 860: 849-860.

[32]Collazo P, Montoliu L, Puigdomènech P, Rigau J. Structure and expression of the lignin O-methyltransferase gene from Zea mays L. Plant Molecular Biology, 1992, 20: 857-867.

[33]Gowri G, Bugos R C, Campbell W H, Maxwell C A, Dixon R A, Division P B, Samuel T, Noble R, Box P O, Oklahoma G G. Stress responses in Alfalfa (Medicago sativa L.). Plant Physiology, 1991: 7-14.

[34]Maury S, Geoffroy P, and Legrand M. Tobacco O-methyltransferases involved in phenylpropanoid metabolism. The different caffeoyl- coenzyme A/5-hydroxyferuloyl-coenzyme A 3/5-O-methyltransferase and caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase classes have distinct substrate spec. Plant Physiology, 1999, 121: 215-224.

[35]Boerjan W, Ralph J, Baucher M. Lignin biosynthesis. Annual Review of Plant Biology, 2003, 54: 519-546.

应用抑制性差减杂交技术筛选锦橙CTV应答基因

Screening of ‘Jincheng’ Orange CTV Response Genes Using Subtractive Suppression Hybridization

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