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作 者 ：段经华1,2，张 琳3，李芳东2，杜红岩2，袁德义3，马履一1*

期 刊 ：西北植物学报 2012年 12期 页码：2374~2382

关键词：枣；MADS-box基因家族；PI基因；抑制消减杂交文库；快速扩增cDNA末端；

Keywords:Ziziphus jujuba Mill., MADS-box families, PI gene, suppression subtractive hybridization library, rapid amplification of cDNA ends,

摘 要 ：以‘金丝4号’枣花蕾和叶片为材料，通过抑制消减杂交文库（SSH文库）的构建、RACE技术和半定量RT-PCR等方法，克隆了枣花发育基因并研究了其时空表达模式，为枣花发育机理研究奠定基础。结果表明：（1）从枣花SSH文库中鉴定出476 bp的花发育B类PISTILLATA（PI）基因的EST序列，命名为ZjMADS1-box；通过RACE技术获得了枣ZjMADS1-box基因的全长cDNA序列。（2）枣ZjMADS1-box基因表现出PI基因的序列结构特征。其全长cDNA长度为957 bp，编码220个氨基酸残基；在氨基酸序列水平上，该基因与巨桉EgM2（Eucalyptus grandis M2）和白千层MqPI（Melaleuca quinquenervia PI）的相似性最高为72.6%，与辣椒CaPI（Capsicum annuum PI）的相似性最低为61.8%；系统进化树分析显示,枣ZjMADS1-box与MqPI和EgM2基因聚在一起；枣ZjMADS1-box基因编码蛋白的等电点为9.75，分子量为25 587.5 Da，属亲水性的非分泌性蛋白。（3）ZjMADS1-box基因在花器官中特异表达,在嫩茎、成熟叶、腋芽等营养器官中不表达;在4月30日和5月8日花蕾中的表达量略高于5月4日、5月12日、5月16日的表达量，但差异不显著。

Abstract:With developing floral buds and leaves as materials,we studied ZjMADS1-box cloning and its expression profiles by construction of a suppression subtractive hybridization (SSH) library,RACE technology,and semi-quantitative RT-PCR.The results are as follows:(1)A 476 bp EST was identified as PISTILLATA (PI) gene related to floral development that was denominated as ZjMADS1-box.Based on the 476 bp sequence information,the full-length cDNA of ZjMADS1-box was successfully obtained by RACE from floral buds of the cultivar ‘Jinsi No.4’.(2)The ZjMADS1-box displays structural characters of PI homologs.The full-length cDNA of ZjMADS1-box is 957 bp,encoding 220 amino acids.On the amino acid sequence level,this gene shows the highest similarity (72.6%) with EgM2 gene of Eucalyptus grandis and MqPI gene of Melaleuca quinquenervia,and the lowest (61.8%) with CaPI gene of Capsicum annuum.ZjMADS1-box clusters with EgM2 and MqPI in phylogenetic tree.The predicted isoelectric point (pI) and molecular weight (Mw) of ZjMADS1-box is 9.75 and 25 587.5 Da,respectively.ZjMADS1-box is predicted to be a kind of hydrophilic and non-secreted proteins.(3)ZjMADS1-box is specifically expressed in floral organs.It is not expressed in vegetative tissues (organs) including tender stems,mature leaves,and axillary buds.In floral buds,expression level of ZjMADS1-box on April 30 and May 8 is higher than that on May 4,May 12,and May 16.The expression level,however,has no remarkable difference between different developing stages.This study will provide a scientific basis for fully clarifying the mechanism of floral development of Z.jujuba.

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