北美鹅掌楸CPP转录因子家族LtTCX2基因的克隆与分析

Cloning and expression analysis of CPP transcription factor family LtTCX2 gene in Liriodendron tulipifera

关键词:  北美鹅掌楸, CPP-like家族, LtTCX2基因, CRC结构域, 组织特异性表达

Key words:  Liriodendron tulipifera, CPP-like family, LtTCX2 gene, CRC domain, tissue specific expression

摘要:

CPP(cystein-richpolycomb-likeproteinor Tesmin/TOS1-like)家族属于成员数目较少的一类转录因子基因家族,含有保守的富含Cystein的CRC结构域,在植物发育进程中,主要参与花发育、细胞分裂、分子进化等。为了探索CPP转录因子家族在北美鹅掌楸花发育中的作用,该文以北美鹅掌楸(Liriodendron tulipifera)为材料,采用RACE技术克隆出1个CPP-like家族基因,命名为LtTCX2,全长2 866 bp。通过NCBI网站在线分析,ORF长2 424 bp,编码了807个氨基酸,含2个保守的TSO1-like CXC结构域,分子量为88 699.25 Da,理论等电点为5.83,不稳定系数为62.38,疏水性平均值为-0.619,预测为亲水性蛋白,非跨膜蛋白,核蛋白,不含信号肽及切割位点。氨基酸比对及系统进化分析结果显示,LtTCX2与其他物种的CPP家族TCX蛋白具有较高的同源性,与亚洲莲(Nelumbo nucifera)的NnTCX2、胡杨(Populus euphratica)的PeTCX2进化关系最近。荧光定量PCR结果显示,LtTCX2基因在叶片中表达量最高,在萼片、花瓣中几乎不表达,表达量由高至低如下:叶片、花芽、雌蕊、雄蕊、茎、根、花瓣、萼片。以上结果说明,LtTCX2属于较古老、保守的一类基因,可为从分子生物学层面研究鹅掌楸属植物系统进化提供一定的理论依据。

Abstract:

CPP(Cystein-rich polycomb-like protein or Tesmin/TOS1-like)protein family are one kind of transcription factors with fewer members and possess a conserved, Cys-rich CRC domains. CPP transcription factor family plays an important role in the plant development of reproductive tissue and cell division. In this study, a CPP-like family gene was cloned by RACE methods from Liriodendron tulipifera named LtTCX2 to explore the function in regulating flower development. The full length of LtTCX2 was 2 866 bp. LtTCX2 contained an open reading frame(ORF)of 2 424 bp, encoding 807 amino acids with two conserved TSO1-like CXC domains by bioinformatics analysis on NCBI site. The molecular weight of protein was 88 699.25 Da, isoelectric point was 5.83 and coefficient of instability was 62.38. LtTCX2 was predicted to be hydrophilic and non-transmembrane nucleoprotein without signal peptide. The amino acid homology sequence and phylogenetic analysis demonstrated that LtTCX2 had high homology with other CPP-like family proteins and was most closely related with NnTCX2 in Nelumbo nucifera and PeTCX2 in Populus euphratica. Real-time quantitative PCR showed that LtTCX2 gene was highly expressed in the leaf and hardly expressed in the sepal and petal. The tissue specific expression order from high to low was that the leaf, flower bud, pistil, stamen, stem, root, petal and sepal. These results suggested that LtTCX2 is a rather conservative gene and provide several help to the phylogenetic evolution of Liriodendron plants from the molecular biology level.