绿竹BoCOBL基因的分子特征及其表达分析

Molecular Characteristics and Expression Analysis of BoCOBL Gene from Green Bamboo (Bambusa oldhamii)

摘要:

为探讨COBRA 相像蛋白(COBL)在竹子细胞发育过程中的作用,采用RT-PCR 和RACE 技术,从绿竹(Bambusa oldhamii)叶片中克隆到1 个COBL 同源基因BoCOBL(GenBank登录号:EU247930),cDNA 全长为1743 bp。序列分析表明,BoCOBL 编码含451 个氨基酸的COBL 蛋白,其N 端具有1 个明显的跨膜螺旋结构,C 端具有1 个糖基磷脂酰肌醇锚定蛋白信号序列,属于糖基磷脂酰肌醇锚定蛋白家族,为典型的膜蛋白。通过构建BoCOBL::GFP 融合表达载体,并转化到烟草(Nicotiana tabacum)悬浮细胞中,表达分析表明BoCOBL::GFP 融合蛋白定位于细胞膜上,而对照GFP 的分布无特异性,证明BoCOBL 基因编码的蛋白为膜蛋白。组织特异性表达分析表明,BoCOBL 基因的表达模式为组成型,在根、茎、叶片和叶鞘中均有表达,但在茎中的表达丰度略低。这为深入研究BoCOBL 基因在竹子中的功能奠定了基础。

关键词:绿竹;COBRA相像蛋白基因;分子特征;亚细胞定位;组织特异性表达

Abstract:

In order to understand the function of COBL similar protein in bamboo, BoCOBL, a COBL homologous gene, was clonned from Bambusa oldhamii by RT-PCR and RACE methods. The full-length cDNA of BoCOBL was 1743 bp (GenBank accession No.: EU247930). Sequence analysis showed that BoCOBL encoded 451 amino acids with a transmembrane helical structure at the N-terminal and a glycosylphosphatidylinositolanchored protein signal sequence at the C-terminal, which indicated that it was a membrane protein, belonging to glycosylphosphatidyl inositol-anchored protein family. The expression vector of BoCOBL::GFP was constructed and transformed into suspension cells of tobacco. The BoCOBL::GFP proteins were mainly located on the cytoplasmic membrane, while the control of GFP proteins was non-specific in the transformed cells. So, it was confirmed that the protein encoded by BoCOBL was a membrane protein. The analysis of tissue specific expression indicated that the expression of BoCOBL was constitutive, with similar level in root, leaf and sheath, while slightly low in stem. These results would be helpful for further study on the function of BoCOBL in bamboo.

Key words:Bambusa oldhamii;COBRA like protein (COBL) gene;Molecular characteristics;Subcellular localization;Tissue specific expression