Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (5): 917-925.doi: 10.3864/j.issn.0578-1752.2012.05.012

• HORTICULTURE • Previous Articles     Next Articles

Molecular Cloning of Waterless-Related Genes in Ponkan Mandarin Using Suppression Subtractive Hybridization

 YANG  Ming, WANG  Ri-Kui, ZHOU  Lian, GE  Wen-Dong, JIAO  Yan-Xiang   

  1. 1.西南大学园艺园林学院，重庆 400715
    2.中国农业科学院柑桔研究所，重庆 400712
    3.西南大学食品科学学院，重庆 400715
  • Received:2011-08-22 Online:2012-03-01 Published:2011-12-21

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Abstract: 【Objective】The aim of this experiment is to reveal the molecular mechanism of the waterless citrus, to explore related genes, and to lay a foundation for the citrus waterless prevention.【Method】A suppression subtractive hybridization library was constructed using cDNA synthesized from RNA extracted from normal pulp as driver and waterless pulp as tester.【Result】 A total of 300 positive clones were selected for sequencing, and a total of 260 EST sequences were obtained. After comparison with GenBank using the online software of the BLAST, 197 ESTs, involving in 52 genes, were found to share considerable homology with known genes while the rest 63 ESTs had low or even no homology with known genes. The expressions of the β-tubulin, senescence-associated protein, cytochrome c, cysteine protease, phosphoenolpyruvate carboxykinase, trafficking protein, aspartic protease precursor, WRKY transcription factor 21 genes were studied by real-time quantitative PCR. The eight genes were significantly up-regulated in waterless pulp. These differentially expressed genes were related to numerical biological processes such as aging, stress-tolerance, chitin and cell wall macromolecule catabolic and proteolysis. 【Conclusion】 Some genes related to waterless were obtained, while the suppression subtractive hybridization library was constructed. These genes reflected the regulation of pulp to waterlessness, and can be used to prevent the disorder of waterlessness as candidate genes.

Key words: Ponkan Mandarin, suppression subtractive hybridization, waterlessness, expressed sequence tags, subtractive library, gene ontology

[1]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.

[2]Von Stein O D, Thies W G, Hormann M. A high throughput screening for rarely transcribed differentially expressed genes. Nucleic Acids Research, 1997, 25: 2598-2602.

[3]Forment J, Gadea J, Huerta L, Abizanda L, Agusti J, Alamar S, Alos E, Andres F, Arribas R, Beltran J P. Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies. Plant Molecular Biology, 2005, 57(3): 375-391.

[4]Yang G P, Ross D T, Kuang W W, Weigel R J. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes. Nucleic Acids Research, 1999, 27(6): 1517-1523.

[5]刘 军. 应用抑制性差减杂交法分离水稻幼穗发育早期特异表达基因. 科学通报, 2000, 45(13): 1392-1397.

Liu J. Suppression subtractive hybridization separated early development of rice panicle-specific genes. Chinese Science Bulletin, 2000, 45(13): 1392-1397.(in Chinese)

[6]朱晓琴, 沈元月, 冯永庆, 秦 岭. 应用抑制性消减杂交分离板栗短雄花序芽变相关基因. 果树学报, 2009, 26(3): 340-343.

Zhu X Q, Shen Y Y, Feng Y Q, Qin L. Application suppressant subtraction hybrid separation Chinese chestnut short male inflorescence bud mutation related genes. Journal of Fruit Science, 2009, 26(3): 340-343. (in Chinese)

[7]张玉刚, 许雪峰, 李天忠, 王 忆, 孔 谨, 韩振海. 抑制性消减杂交分离苹果缺铁诱导的相关基因. 园艺学报, 2007(3): 555-560.

Zhang Y G, Xu X F, Li T Z, Wang Y, Kong J, Han Z H. Apple suppression subtractive hybridization separation of iron deficiency-induced genes. Acta Horticulturae Sinica, 2007(3): 555-560. (in Chinese)

[8]高 雪, 李正国, 樊 晶, 杨迎伍. 奉节脐橙果皮褐变差减文库的构建及初步分析. 植物生理与分子生物学学报, 2007, 33(1): 71-76.

Gao X, Li Z G, Fan J, Yang Y W. Fengjie navel orange peel browning subtractive library construction and preliminary analysis. Journal of Plant Physiology and Molecular Biology, 2007, 33(1): 71-76. (in Chinese)

[9]Sung K C, Ji U J, Kung H K, Kyu S S, Kwang W J, Min K Y, Sung H O, Yong S C, Heung G H, Hae C C, Huhn P M, Jeong S S. Identification of genes induced in wound treated wild rice(Oryza minuta). Molecules and Cell, 2004, 17(2): 230-236.

[10]程春振, 朱世平, 吴 波, 阳佳位,贝学军,马岩岩, 钟广炎.紫外线照射对梁平柚果皮基因表达的影响. 园艺学报, 2011, 38(5): 859-866.

Cheng C Z, Zhu S P, Wu B, Yang J W, Bei X J, Ma Y Y, Zhong G Y. Effect of UV-B radiation on gene expression in peels of ‘Liang ping’pommelo. Acta Horticulturae Sinica, 2011, 38(5): 859-866. (in Chinese)

[11]叶庆亮, 江 东, 彭爱红. ‘岩溪晚芦’椪柑果皮与果肉差减cDNA文库的构建与初步分析. 园艺学报, 2009, 36(7): 967-974.

Ye Q L, Jiang D, Peng A H. Construction of a subtractive cDNA library between fruit epicarp and pulp of PonKan Mandarin and primary analyses of the sequences. Acta Horticulturae Sinica, 2009, 36(7): 967-974. (in Chinese)

[12]Hisada S, Moriguchi T, Hidaka T, Koltunow A M, Akihama T, Omura M. Random sequencing of sweet orange cDNA library derived from young seeds. Journal of the Japanese Society for Horticultural Science, 1996, 65: 487-495.

[13]Hisada S. Expressed sequence tags of citrus fruit during rapid cell development phase. Journal of the Japanese Society for Horticultural Science, 1997, 122: 808-812.

[14]Hisada S, Akihama T, Endo T, Moriguchi T, Omura M, Moriguchi T. Refinament of cDNA clone expression analysis in random sequencing from the rapid cell development phase of citrus fruit. Journal of Plant Physiology, 1999, 155: 699-705.

[15]Moriguchi T, Kita M, Hisada S, Endo-Inagaki T, Omura M. Characterization of gene repertoires at mature stage of citrus fruits through random sequencing and analysis of redundant metallothionein- like genes expressed during fruit development.Gene, 1998, 211: 221-227.

[16]Shimada T, Hirbayashi T, Endo T, Fujii H, Kita M, Omura M. Isolation and characterization of the somatic embryogenesis receptor-like kinase gene homologue from citrus unshiu maru. Acta Horticulturae Sinica, 2004, 103: 233-238.

[17]Kita M, Endo-Inagaki T, Moriguchi T, Omura M. cDNA catalogs expressed in albedo of citrus fruit, a comparative analysis of cDNA libraries from pulp and albedo of Satsuma mansdarin. Acta Horticulturae Sinica, 2000, 521: 179-183.

[18]Gao X, Li Z G, Fan J, Yang Y W. Screening and expression of differentially expressed genes for peel pitting of citrus fruit. Acta Horticulturae Sinica, 2003, 218(1): 65-70.

[19]Liu Q, Zhu A D, Chai L J, Zhou W J, Yu K Q, Ding J, Xu J, Deng X  X. Transcription analysis of a spontaneous mutant in sweet orange during fruit development. Journal of Experimental Botany, 2009, 60(3): 801-813.

[20]Kost B, Mathur J, Chua N H. Cytoskeleton in plant development. Current Opinion in Plant Biology, 1999, 2: 462-470.

[21]Kopczak S, Hass N, Hussey P, Silflow C D, Snustad D P. The small genome of Arabidopsis contains at least six expressed alpha-tubulin genes. The Plant Cell, 1992, 4: 539-547.

[22]Snustad D, H N, Kopczak S，Snustad D P. The small genome of Arabidopsis contains at least nine expressed beta-tubulin genes. The Plant Cell, 1992, 4: 549-556.

[23]Carpenter J, Ploense S, Snustad D P, Silflow C D. Preferential expression of an α- tubulin gene of Arabidopsis in pollen. The Plant Cell, 1992, 4: 557-571.

[24]Chu B, Wilson T, McCune Z C, John V C. TUB1 and TUB8, of Arabidopsis exhibits largely nonoverlapping patterns of expression. Plant Molecular Biology, 1998, 37: 785-790.

[25]王中华, 应天雷, 黄仲贤. 细胞色素c突变研究进展. 化学通报，2010, 1: 31-35.

Wang Z H, Ying T L, Huang Z X. Research progress of cytochrome c mutation. Chemistry, 2010, 1: 31-35. (in Chinese)

[26]Srinivasa M S，Manzoor A, Teresa F A, Gerald L, Emad S A. Molecular ordering of the Fas-apoptotic pathway: The Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases. Proceedings of the National Academy of Sciences of the United States of America, 1996, 93: 14486-14491.

[27]Gonzalez M C, Sanchez R, Cejudo F J. Abiotic stresses affecting water balance induce phosphoenolpyruvate carboxylase expression in roots of wheat seeding. Planta, 2003, 216(6): 985-1002.

[28]Conceicao E, Nuno L, Rosa R, Rui M, Brito M. Euclides P, Maria C, Pesroso D L, Carlos F. The saposin-like domain of the plant aspartic proteinase precursor is a potent inducer of vesicle leakage. The Journal of Biological Chemistry, 2000, 49(8): 18190-18196.

[29]Tetsu K, Kenji Y, Nagako H, Maki K, Mikio N, Ikuko H N. Vacuolar processing enzyme is up-regulated in the lytic vacuoles of vegetative tissues during senescence and under various stressed conditions. The Plant Journal, 1999, 19(1): 43-53.

[30]张立勇, 赵晓航, 吴 旻. 膜联蛋白Ⅰ的结构和功能. 生物化学与生物物理进展, 2002, 29(4): 514-517.

Zhang L Y, Zhao X H, Wu M. AnnexinⅠstructure and function, Progress of Biochemistry and Biophysics, 2002, 29(4): 514-517. (in Chinese)

[31]Hao Y J, Hiroyasu K, Chikako H, Kazuyoshi N, Takaya M. Expression of arginine decarboxylase and ornithine decarboxylase genes in apple cells and stressed shoots. Journal of Experimental Botany, 2005, 56(414): 1105-1112.
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