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【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】 To explore the molecular mechanism of CTV response in Citrus tristeza virus (CTV) variety and screen and analyze CTV response genes. 【Method】 The grafted CTV virulent strain TRL514 was used as the experimental host (Tester), and CTV-negative broccoli leaves grafted with avirulent shoots were used as the driver to construct CTV-infected broccoli positive Subtraction library. 【Result】 A total of 850 positive clones were randomly selected and sequenced. Among them, 742 were sequenced successfully, and 692 ESTs were obtained after removing the vector fragment and linker sequence. All EST sequences were mapped to the predicted genomic transcripts of citrus, involving a total of 137 citrus genes. The functionally classified and KEGG analyzes of these specifically expressed genes were performed using BLAST2GO. The results showed that under the influence of CTV infection, the genes related to energy metabolism of Jincheng were significantly up-regulated, and ESTs related to photosynthetic organs such as carbon fixation, nitrogen metabolism, starch and sucrose metabolism were higher. In addition, there are many genes related to anti-retrograde defense and metabolic pathways that are closely related to the synthesis of plant defensins, such as phenylalanine synthesis, phenylpropane metabolism, and carotenoid synthesis. 【Conclusion】 Due to the infection of CTV, the basic metabolism of the susceptible variety CTV changed obviously. It is necessary to maintain its own growth and development by enhancing energy metabolism and enhancing self-defense ability.