利用SSH分离TDZ诱导金钗石斛成花相关基因

园艺学报 ›› 2013, Vol. 40 ›› Issue (8): 1591-1599.

利用SSH分离TDZ诱导金钗石斛成花相关基因

闻真珍，张娥，刘运权，刘伟*

（华南农业大学生命科学学院，广州 510642）

收稿日期:2013-01-28 出版日期:2013-08-25 发布日期:2013-08-25

Primary Screening of Differentially Expressed Genes During TDZ Induced Floral Initiation with SSH in Dendrobium nobile

WEN Zhen-zhen，ZHANG E，LIU Yun-quan，and LIU Wei*

Received:2013-01-28 Online:2013-08-25 Published:2013-08-25

摘要/Abstract

摘要： 以金钗石斛（Dendrobium nobile Lindl.）腋芽为材料，利用抑制消减杂交（SSH）技术构建了TDZ处理后金钗石斛腋芽的正向和反向SSH文库。通过PCR和反向Northern杂交验证后，得到的正向文库的314个阳性克隆和反向文库的59个克隆测序后经过聚类，最终得到39条受TDZ正向调控的EST序列，以及21条受其反向调控的EST序列。对其中某些差异基因进行半定量RT-PCR，结果显示这些基因的表达受TDZ的影响。利用Blastn和Blastx进行比对分析，其中20个没有同源序列，属于未知功能基因。其余序列的同源基因编码的产物参与包括光合作用、合成代谢、转录调控等多种生物学过程。在反向文库中分离到1个MADS-box基因的同源序列和1个Sos同源基因，而正向文库中存在多个反转录转座子，这些结果表明TDZ可以影响开花过程中的某些转录因子和信号基因的表达，进而促进金钗石斛开花。

关键词: 金钗石斛, 开花, 细胞分裂素, 抑制消减杂交（SSH）, 反转录转座子

Abstract: A forward and a reverse suppression subtractive cDNA library were constructed，using cDNAs from the TDZ-treated and non-treated axillary buds of Dendrobium nobile Lindl. A total of 314 and 59 recombinant clones were identified from the forward and reverse SSH library respectively，by PCR and reverse Northern blotting. All of these clones were sequenced and assembled，finally 39 ESTs from the forward library and 21 ESTs from the reverse library were obtained. Effects of TDZ on the transcription of some ESTs were confirmed by semi RT-PCR. Of these sequences，twenty genes were not homologious to the reported genes in GenBank，the rest were classified into photosynthesis，biosynthesis，metabolism or regulatory function. A sequence homologious to MADS-box genes and a homology of Sos were identified from the reverse library，and several retrotransposons were characterized from the forward library，these results indicated that TDZ could promote the flowering of D. nobile，by affecting the transcriptions of some transcription factors and signal genes.

Key words: Dendrobium nobile Lindl., flowering；cytokinin, suppression subtractive hybridization（SSH）, retrotransposon

闻真珍，张娥，刘运权，刘伟*. 利用SSH分离TDZ诱导金钗石斛成花相关基因[J]. 园艺学报, 2013, 40(8): 1591-1599.

WEN Zhen-zhen，ZHANG E，LIU Yun-quan，and LIU Wei*. Primary Screening of Differentially Expressed Genes During TDZ Induced Floral Initiation with SSH in Dendrobium nobile[J]. ACTA HORTICULTURAE SINICA, 2013, 40(8): 1591-1599.

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