关键词: 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cDNA为测试方, 以脱毒的‘不知火’实生苗叶片组织的cDNA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cDNA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。

Abstract:

In order to elucidate the molecular mechanism of the response of citrus to Citrus tristeza virus infection at late stage，a cDNA library was constructed by suppression subtractive hybridization with the RNAs from the leaves of citrus cultivar‘Shiranui’. Two hundred and ten positive clones were picked out randomly from library for sequencing. A total 192 valid ESTs were obtained. Clustering analysis indicated that 77 of them were non-repetitive sequences. Based on GO and KEGG analysis of these ESTs sequences from citrus genome database，the main functions of these ESTs were classified related to disease defense，stress，metabolism，transcription，transport and protein fate. Real-time RT-PCR analysis indicated that expression of 4 genes，including chitinase，jasmonate zim domain 1（JAZ1），calmodulin binding protein，caffeate O-methyltransferase were significantly up-regulated when citrus was infected by Citrus tristeza virus，indicating that these genes may be involved in the resistant response to Citrus tristeza disease. In this paper，we discuss the up-regulation of JAZ1 gene may indicate that the induction of systemic resistance in response to Citrus tristeza disease at late stage of virus infection is inhibited.

Key words: citrus, Citrus tristeza disease, suppression subtractive hybridization（SSH）, responsive gene