番茄COBRA基因克隆、表达模式及生物信息学分析

COBRA作为一种重要的胞外糖基磷脂酰肌醇(GP-I)锚定蛋白,影响植物细胞壁中纤维素含量及细胞的定向伸长。目前已有多个拟南芥、玉米及水稻的COBRA基因的突变体被研究。而有关番茄COBRA基因克隆的研究尚未见报道。本研究利用RT-PCR技术克隆了一个假定编码番茄COBRA蛋白的SlCOBRA基因,并在GenBank注册(JN398667)。序列测定和分析表明,该序列由6个外显子组成,编码444个氨基酸残基;氨基酸序列中存在COBRA蛋白的CCVS保守基序,N端的跨膜信号肽及C-末端的疏水性尾部和GPI锚定ω-位点。系统进化分析表明番茄SlCOBRA与拟南芥AtCOB具有80%氨基酸序列同源性,聚在一个分支上。Real-time PCR分析番茄各个组织中COBRA基因的表达结果表明番茄COBRA为组成型表达,在营养器官(根、茎、叶)中的表达量高于花和果实,尤其在成熟的果实中(从转色期到红熟期)表达量明显减少。

Cloning,Expression Pattern and Bioinformation Analyses of COBRA Gene in Tomato(Solanum lycopersicum)

COBRA is a member of plant-specific glycosylphosphatidylinositol(GPI)-anchored protein family and plays an important role in controlling cellulose content of cell wall and orientation of cell expansion.Although studies on COBRA mutants from Arabidopsis,Oryza sativa and Zea mays have been extensively performed,the function of ortholog of COBRA in tomato remains largely unknown.In this study,a full-length cDNA sequence highly homologous with other plant COBRA genes was identified by RT-PCR from tomato seeding,and named as SlCOBRA(GenBank Accession No.JN398667).Results of sequence analyses showed that SlCOBRA is composed of six exons encoding a 444-amino acid protein,containing conserved features of the COBRA protein,such as a CCVS motif,an N-terminal signal peptide sequence for secretion,a highly hydrophobic C terminus,and specific features around the ω-site required for processing.Phylogenetic analysis revealed that the deduced amino acid sequence of SlCOBRA shows 80% identity with that of Arabidopsis AtCOB,thereby being organized in the same clade.Meanwhile,the expression pattern of tomato COBRA was revealed by real-time PCR.The results showed that tomato COBRA exhibits higher expression levels in roots,shoots,flower and fruits,but only during the green stages of tomato fruit development,prior to visible signs of ripening.At the start of the breaker stage,the levels of tomato COBRA mRNA decline dramatically.