Identification of differentially expressed genes in 4-day axolotl limb blastema by suppression subtractive hybridization
Identification of differentially expressed genes in 4-day axolotl limb blastema by suppression subtractive hybridization
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DOI：
10.1007/bf03168233
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发表时间：
2008-03-01
影响因子：
3.4
通讯作者：
Kornel, R.
中科院分区：
文献类型：
--
作者：
Gorsic, M.;Maidic, G.;Kornel, R.
本研究的目的是利用抑制减法杂交（SSH）技术鉴定美西螈（Ambystoma mexicanum）截肢后4天后肢再生过程中的上调基因。选择了大约400个在再生胚组织中含有上调基因的克隆进行序列分析。对NCBI非冗余数据库和ambystoma EST数据库进行BLAST同源性检索，发现102个克隆与GenBank中已知序列同源，具有注释功能，31个为已知基因，不具有已知功能，74个为新基因，72个属于线粒体序列。qRT-PCR分析证实Hmox1、Orc4L、NO、Fen-1、Mcm7和Mmp3/10a的差异表达。在所有基因中，只有Mmp3/10a先前被描述为参与肢体再生。其他重要的鉴定基因属于细胞周期调节因子组（Orc4L, Nasp， Skp1A和Mcm7，后者可能是一个增殖标记），参与蛋白质合成和运输的基因（Sec63, Srp72, Sara2）和V- atp酶泵。我们发现的新基因可能对再生肢体的胚泡形成过程和细胞增殖的开始很重要。
The goal of our study was the identification of up-regulated genes during axolotl (Ambystoma mexicanum) hindlimb regeneration 4 days after amputation using suppression subtractive hybridization (SSH). Approximately 400 clones that harbored upregulated genes in regenerating blastema tissue were selected for sequence analysis. A BLAST homology search against NCBI non-redundant database and an ambystoma EST database revealed 102 clones that showed homology to known sequences in GenBank with annotated function, 31 were known genes without known function, 74 were novel and 72 belonged to mitochondrial sequences. Differential expression of Hmox1, Orc4L, NO, Fen-1, Mcm7 and Mmp3/10a was confirmed using qRT-PCR analysis. Among all genes, only Mmp3/10a has been previously described as involved in limb regeneration. Other important identified genes belong to the group of cell cycle regulators (Orc4L, Nasp, Skp1A and Mcm7, the latter being a possible proliferative marker), those involved in protein synthesis and transport (Sec63, Srp72, Sara2) and V- ATPase pump. The novel genes we identified might be important for the process of blastema formation and the onset of cell proliferation in a regenerating limb.