小麦细胞色素P450(TaP450)基因的克隆与序列分析

Cloning and Sequence Analysis of a Cytochrome P450(TaP450 )Gene from Wheat(Triticum aestivum L.)

细胞色素P450是一种多功能氧化酶,在植物体内担当着生物合成、代谢解毒以及抗逆等重要功能。本研究采用RACE方法从普通小麦中同源克隆到一个新的P450基因,命名为TaP450(Genbank No.KJ541960)。该基因基因组序列全长为2 643bp,含有2个外显子和1个内含子,cDNA全长为2 033bp,含有一个1 557bp的开放读码框,推导蛋白含518个氨基酸;序列分析发现其具有保守的P450结构域,但该蛋白与已报道的小麦P450蛋白序列有较大差异,表明它是小麦P450家族的新成员;蛋白结构特征分析发现,该蛋白的分子量为57.092kD,等电点为8.63,N端具有一个含29个氨基酸的跨膜结构和一个含22个氨基酸的信号肽,亚细胞定位分析表明该蛋白属于分泌蛋白。其在小麦叶片、茎、根与种子中均有表达,但在叶片和茎中表达量最高;在胁迫处理下,该基因的表达量均上调,且在盐胁迫处理下上调尤为显著,表明该基因与盐胁迫密切相关。

Cytochrome P450is a multifunctional enzyme,playing important roles in biosynthesis,metabolism detoxification and stress resistance in plants.Here,a P450gene(TaP450,Genbank No.KJ541960)were cloned using homologous-based PCR combined with RACE method from common wheat.The genomic length of the cloned TaP450 was 2 643bp with two exons and one intron.The full length cDNA was 2 033bp,containing an open reading frame of 1 557bp.The deduced protein had 518amino acids(AA).Protein sequence analysis found that it had the conserved P450domain,but its sequence composition was different from other reported wheat P450genes,which indicated that it was a new member of P450family in wheat.The predicted molecular weight of this protein wasabout 57.092kD with the isoelectric point of 8.63.A transmembrane structure of 29amino acids and a signal peptide of 22amino acids were also found in its N-terminus.Semi-quantitative RT-PCR analysis showed that this gene expressed in all the tested organs,including leaves,stems,roots and seeds,while the highest expression level was found in leaves and stems.Furthermore,TaP450 was found to be up-regulated significantly under different stresses,and the highest expression level was observed under salt stress.These results indicated clearly that TaP450 might play a vital role in response to abiotic stresses,especially to salt stress in wheat.The current study isolated a new P450gene from common wheat,which enriched the abundance of wheat P450gene family and also shed light on revealing the biological function of this P450gene in wheat.