中国农业科学2012，Vol.45Issue(5)：917-925,9.DOI:10.3864/j.issn.0578-1752.2012.05.012
应用抑制性差减杂交技术分离椪柑枯水相关基因
Molecular Cloning of Waterless-Related Genes in Ponkan Mandarin Using Suppression Subtractive Hybridization
摘要
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 P-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.关键词
椪柑/抑制性差减杂交/枯水/表达序列标签/差减文库/聚类分析Key words
Ponkan Mandarin/ suppression subtractive hybridization/ waterlessness/ expressed sequence tags/ subtractive library/ gene ontology引用本文复制引用
杨明,王日葵,周炼,葛文东,焦雁翔..应用抑制性差减杂交技术分离椪柑枯水相关基因[J].中国农业科学,2012,45(5):917-925,9.基金项目
重庆市自然科学基金项目(CSTC,2010BB1154)、重庆市科技攻关项目(CSTC,2011AC1023)、国家公益性行业(农业)科研专项(201203034) （CSTC,2010BB1154）
