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

作　　者：程春振[1,2] 曾继吾[2] 贝学军[1,2] 吴波[1,2] 阳佳位 张永艳[1,2] 姜波[2] 朱世平[1] 闫树堂[1] 钟云[2] 钟广炎[2] 

机构地区：[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

出　　处：《中国农业科学》2013年第16期3413-3423,共11页Scientia Agricultura Sinica

基　　金：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)

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。【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.

关 键 词：柑橘 抑制性差减杂交技术 差异表达基因 CTV 

分 类 号：S666.2[农业科学—果树学]