棉纤维细胞色素单加氧酶基因GhCYP714A1促进活性氧的累积
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摘要
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 H_2O_2 accumulation in leaves.These results suggested that GhCYP714A1 may be involved in fiber development by promoting the production of ROS.
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 H_2O_2 accumulation in leaves.These results suggested that GhCYP714A1 may be involved in fiber development by promoting the production of ROS.
引文
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[2]钱雯婕,王斐,李鸿彬,等.棉花单脱氢抗坏血酸还原酶基因的克隆及原核表达[J].西北农业学报,2012,21(5):118-122.Qian W J,Wang F,Li H B,et al.Cloning and prokaryotic expression of a cotton monodehydroascorbate reductase gene[J].Acta Agriculturae Boreali-occidentalis Sinica,2012,21(5):118-122.
[3]Hartweck L M and Olszewski N E.Rice Gibberellin insensitive dwarf1 is a gibberellin receptor that illuminates and raises questions about Ga signaling[J].The Plant Cell,2006,18(2),278-282.
[4]Fu X and Harberd N P.Auxin promotes Arabidopsis root growth by modulating gibberellin response[J].Nature,2003,421(6924):740-743.
[5]Bennett T,Sieberer T,Willett B,et al.The Arabidopsis MAX pathway controls shoot branching by regulating auxin transport[J].Curr Biol,2006,16(6):553-563.
[6]Qin YM,Hu CY,Zhu YX.The ascorbate peroxidase regulated by H2O2and ethylene is involved in cotton fiber cell elongation by modulating ROS homeostasis[J].Plant Signaling&Behavior,2008,3(3):194-196.
[7]Gialvalis S,Seagull R W.Plant hormones alter fiber initiation in unfertilized,cultured ovules of Gossypium hirsutum[J].The Journal of Cotton Science,2001,5(4):252-258.
[8]Xiao Y H,Meng H Y,Hou L,et al.Direct amplification of introncontaining hairpin RNA construct from genomic DNA[J].Biotechniques,2006,41(2):548,550,552.
[9]Zhang Y Y,Zhang B C,Yan DW,et al.Two Arabidopsis cytochrome P450 monooxygenases,CYP714A1 and CYP714A2,function redundantly in plant development through Gibberellin deactivation[J].Plant J,2011,67(2):342-353.
[10]蒋建雄,张天真.利用CTAB-酸酚法提取棉花组织总RNA[J].棉花学报,2003,15(3):166-167.Jiang J X,Zhang T Z.Extraction of total RNA in cotton tissues with CTAB-acidic phenolic method[J].Cotton Science,2003,15(3):166-167.
[11]Bellincampi D,Dipierro N,Salvi G,et al.Extracellular H2O2induced by oligogalacturonides is not involved in the inhibition of the auxinregulated rolb gene expression in tobacco leaf explants[J].Plant Physiology,2000,122:1379-1385.
[12]Kende H,Zeevaart J.The five“classical”plant hormones[J].Plant Cell,1997,9(7):1197-1210.
[13]Foreman J,Demidchik V,Bothwell J H,et al.Reactive oxygen species produced by NADPH oxidase regulate plant cell growth[J].Nature,2003,422(6930):442-446.
[14]Mittler R,Vanderauwera S,Gollery M,et al.Reactive oxygen gene network of plants[J].Trends Plant Sci,2004,9(10):490
[15]Schopfer,P.Hydroxyl radical-induced cellwall loosening in vitro and in vivo:implications for the control of elongation growth[J].Plant J,2001,28(6):679-688.