棉纤维细胞色素单加氧酶基因GhCYP714A1促进活性氧的累积

Overexpression of a Gh CYP714A1 gene encoding cytochrome P450 gene promotes the H_2O_2 accumulation in transgenic tobacco plant

本研究利用RT-PCR技术从陆地棉纤维组织中克隆得到了1个棉花细胞色素P450基因(Cytochrome P450714A1)的全长c DNA序列,将该基因命名为GhCYP714A1。序列分析发现,该cDNA包含1563 bp的完整开放读码框,编码521个氨基酸的蛋白质,理论分子质量为57.31 kDa;氨基酸序列生物信息学分析显示,Gh CYP714A1蛋白具有跨膜结构域和多个蛋白结合位点;进化树分析结果表明,棉花Gh CYP714A1与禾草SiCYP714B1在进化上亲缘关系上较近;半定量RT-PCR的组织表达特异性分析表明GhCYP714A1基因与纤维突起形成发育有关。本研究构建了pET28a-GhCYP714A1原核表达载体并进行体外诱导表达,获得分子质量约为57.31 kDa的重组蛋白。将GhCYP714A1基因转化烟草验证其参与活性氧产生的功能,与非转基因的野生型烟草相比,转基因烟草叶片中具有较高的H_2O_2累积。本实验结果为深入研究该基因在棉纤维发育中的作用奠定了基础。

The full-length cDNA of a cytochrome P450 gene（Cytochrome P450 714A1,GhCYP714A1） was cloned from Upland cotton fiber tissues through RT-PCR method.Sequence analysis showed that the GhCYP714A1 cDNA contains a 1563 bp open reading frame（ORF）,which coding a putative protein with theoretical molecular weight of 57.31 kDa and 521 amino acid residues.Bioinformatics analysis demonstrated that GhCYP714A1 protein contains several protein binding sites and a transmembrane functional domain.Phylogenetic tree analysis showed that GhCYP714A1 has close relationship with SiCYP714B1 protein.Tissue-specific expression patterns assay by semi-quantitative RT-PCR showed that GhCYP714A1 was expressed during the initiation and elongation stages of fiber development.The prokaryotic expression vector pET28a-GhCYP714A1 was constructed and transformed into E.coli to induce expression.As a result,a 57.31 kDa recombinant protein was obtained.GhCYP714A1 gene was transformed into tobacco to explore its function in ROS generation.Compared with wild-type tobacco,the transgenic tobacco plants displayed a higher H2O2 accumulation in leaves.These results suggested that GhCYP714A1 may be involved in fiber development by promoting the production of ROS.