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为了研究柑橘衰退病晚期应答的分子机理,利用经典的抑制差减杂交技术,以感染柑橘衰退病3年后的‘不知火’杂柑实生苗叶片组织的c DNA为测试方,以脱毒的‘不知火’实生苗叶片组织的c DNA为驱动方进行差减杂交,构建了柑橘衰退病诱导的正向抑制差减c DNA文库。随机挑取此文库中210个阳性克隆进行测序,获得了192条有效序列。将这些EST序列聚类拼接后,共获得77条非重复序列。对这些EST进行GO和KEGG分析,发现这些EST的功能主要与胁迫及防御、代谢、转录、转运、蛋白命运等途径相关。用实时定量RT-PCR验证了其中4个基因:几丁质酶基因、茉莉酸响应基因的转录抑制因子1基因(JAZ1)、钙调素结合蛋白基因、咖啡酸—氧甲基转移酶基因,结果表明在衰退病的诱导下,‘不知火’杂柑叶片中这4个基因的表达量均有明显提高,说明这些基因可能参与了‘不知火’杂柑叶片晚期衰退病应答。JAZ1基因的上调表达表明可能宿主衰退病晚期应答过程中的诱导系统抗性受到了抑制。
In order to study the molecular mechanism of the late response to citrus recession, the classical suppression subtractive hybridization (SSH) technique was used to test the c DNA of the leaf tissue of Citrus unshiucae infected seedlings three years after the citrus recession, I do not know the fire ’seedling leaf tissue c DNA driven by the subtractive hybridization, the construction of citrus recession-induced positive suppression subtractive c DNA library. A total of 210 positive clones from this library were randomly selected for sequencing and 192 validated sequences were obtained. After clustering these EST sequences, 77 non-repetitive sequences were obtained. GO and KEGG analysis of these ESTs showed that the functions of these ESTs were mainly related to stress and defense, metabolism, transcription, transport and protein fate. Four genes were identified by real-time quantitative RT-PCR: chitinase, JAZ1, calmodulin-binding protein, caffeic-oxyltransferase gene, The results showed that the expression of these four genes was significantly increased in the leaves of Citrus unshiuifeng under the induction of the recessive disease, indicating that these genes may be involved in the late-stage recession response of Citrus unshiucae leaves. Up-regulated expression of JAZ1 gene indicates that induction of systemic resistance during the late phase of the disease may be inhibited.