应用抑制性差减杂交技术分离椪柑枯水相关基因

doi:10.3864/j.issn.0578-1752.2012.05.012

摘要/Abstract

摘要： 【目的】探索椪柑枯水分子机理，寻找柑橘枯水应答基因，为柑橘枯水防治奠定基础。【方法】利用抑制性差减杂交技术以椪柑正常果肉cDNA作为Driver，以枯水果肉cDNA作为Tester构建正向差减文库。【结果】挑选了300个有效克隆进行测序分析，260个测序成功。经BLASTx比对分析，197条表达序列标签（ESTs）找到了分属于52个基因的同源序列；63条ESTs同源性较低或没有同源性。对文库中编码β-微管蛋白、衰老相关蛋白、细胞色素c、半胱氨酸蛋白酶、磷酸烯醇式丙酮酸羧激酶、转运蛋白、天冬氨酸蛋白酶前体、WRKY转录因子21的基因进行实时定量RT-PCR验证，结果显示这8个基因均明显上调表达。这些差异性表达基因主要涉及衰老、抗逆防御、几丁质和细胞壁代谢、蛋白降解等过程。【结论】通过构建椪柑果肉枯水正向文库，得到一些与枯水相关的基因，这些基因反映了果实对枯水的调控情况，可作为今后防治枯水病害的候选基因。

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

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

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.

YANG Ming, WANG Ri-Kui, ZHOU Lian, GE Wen-Dong, JIAO Yan-Xiang. Molecular Cloning of Waterless-Related Genes in Ponkan Mandarin Using Suppression Subtractive Hybridization[J]. Scientia Agricultura Sinica, 2012, 45(5): 917-925.

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