甘蔗脂氧合酶基因ScLOX1的克隆与表达分析
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摘要
LOX属于脂氧合酶超家族(lipoxygenasesuperfamily),是脂肪氧化途径的重要因子,广泛参与植物生长发育的调节和对外界刺激的抵御。本研究基于甘蔗(Saccharumspp.)转录组数据库,通过RT-PCR技术,首次从新台糖22号(ROC22)蔗芽中克隆获得ScLOX1基因(GenBank登录号为MK106188)的cDNA全长序列。生物信息学分析显示,ScLOX1基因cDNA序列长度为2813 bp,开放读码框全长2664 bp,编码887个氨基酸,其编码蛋白的理论等电点为6.23,不稳定系数为39.77,亲水性平均值为?0.437,无信号肽和跨膜结构,但含有PLAT_LH2和Lipoxygenase活性位点,与高粱(Sorghum bicolor) LOX (XP_002466613.1)的氨基酸序列相似性高达95.96%。预测ScLOX1基因的编码蛋白为酸性稳定亲水性非分泌蛋白,属于type I类非传统9-LOX。qPT-PCR分析结果显示,ScLOX1基因在蔗芽组织中特异性表达。接种甘蔗黑穗病菌(Sporisorium scitamineum)后, ScLOX1基因的表达量在抗病品种崖城05-179中短暂上升,但在感病品种ROC22中显著下降。分别对瞬时表达ScLOX1基因的本氏烟(Nicotiana benthamiana)植株叶片接种烟草茄病镰刀菌蓝色变种(Fusarium solani var. coeruleum)和青枯菌(Ralstonia solanacearum),表型观察、3,3’-二氨基联苯胺(3,3’-diaminobenzidine,DAB)染色和烟草免疫相关基因的表达情况分析显示,ScLOX1基因的过表达能够增强本氏烟对烟草茄病镰刀菌蓝色变种的防御,但对烟草青枯菌的作用与对照相比无明显差异。研究还发现,ScLOX1基因的表达受茉莉酸甲酯和水杨酸抑制下调,但受脱落酸、氯化钠和聚乙二醇诱导上调。以上结果为深入研究甘蔗ScLOX1基因的功能提供参考资料。
LOX, which belongs to the lipoxygenase superfamily, is an important factor for fat oxidation and widely involved in the regulation of plant growth and development and the resistance to external stimuli. In this study, based on sugarcane(Saccharum spp.) transcriptome database, we first cloned a full-length c DNA sequence of ScLOX1 gene(GenBank accession number:MK106188) from ROC22 bud by RT-PCR. Bioinformatics analysis showed that the cDNA length of ScLOX1 gene was 2813 bp which had a 2664 bp length of open reading frame, encoding 887 amino acids. The theoretical isoelectric point, instability coefficient, and average hydrophilicity of the ScLOX1 protein were 6.23, 39.77, and ?0.437, respectively. There were no signal peptide and transmembrane structure, but the PLAT_LH2 and lipoxygenase active sites in Sc LOX1 protein. The similarity of amino acid sequences between ScLOX1 and Sorghum bicolor LOX(XP_002466613.1) was 95.96%. The protein encoded by ScLOX1 gene was predicted to be an acid-stable, hydrophilic, and non-secreted protein which belongs to the type I non-traditional9-LOX. qRT-PCR results showed that ScLOX1 was specifically expressed in sugarcane bud tissue. The expression level of ScLOX1 gene was transiently increased in the smut-resistant sugarcane variety Yacheng05-179 but significantly decreased in the smut-susceptible sugarcane variety ROC22 after inoculated with Sporisorium scitamineum. When leaves of Nicotiana benthamiana were transiently overexpressed ScLOX1 gene and inoculated with tobacco pathogens Fusarium solani var. coeruleum and Ralstonia solanacearum, respectively, the results of phenotypic observation, 3,3'-diaminobenzidine(DAB) staining and expression analysis of tobacco immune-related genes revealed that the overexpression of ScLOX1 gene could enhance the defense of N.benthamiana to the F. solani var. coeruleum, but had no significant difference with the control on the defense effect against R.solanacearum. In addition, the expression level of ScLOX1 was down-regulated by methyl jasmine and salicylic acid, but up-regulated by abscisic acid, sodium chloride and polyethylene glycol. The above results provide references for further study on the function of sugarcane ScLOX1 gene.
LOX, which belongs to the lipoxygenase superfamily, is an important factor for fat oxidation and widely involved in the regulation of plant growth and development and the resistance to external stimuli. In this study, based on sugarcane(Saccharum spp.) transcriptome database, we first cloned a full-length c DNA sequence of ScLOX1 gene(GenBank accession number:MK106188) from ROC22 bud by RT-PCR. Bioinformatics analysis showed that the cDNA length of ScLOX1 gene was 2813 bp which had a 2664 bp length of open reading frame, encoding 887 amino acids. The theoretical isoelectric point, instability coefficient, and average hydrophilicity of the ScLOX1 protein were 6.23, 39.77, and ?0.437, respectively. There were no signal peptide and transmembrane structure, but the PLAT_LH2 and lipoxygenase active sites in Sc LOX1 protein. The similarity of amino acid sequences between ScLOX1 and Sorghum bicolor LOX(XP_002466613.1) was 95.96%. The protein encoded by ScLOX1 gene was predicted to be an acid-stable, hydrophilic, and non-secreted protein which belongs to the type I non-traditional9-LOX. qRT-PCR results showed that ScLOX1 was specifically expressed in sugarcane bud tissue. The expression level of ScLOX1 gene was transiently increased in the smut-resistant sugarcane variety Yacheng05-179 but significantly decreased in the smut-susceptible sugarcane variety ROC22 after inoculated with Sporisorium scitamineum. When leaves of Nicotiana benthamiana were transiently overexpressed ScLOX1 gene and inoculated with tobacco pathogens Fusarium solani var. coeruleum and Ralstonia solanacearum, respectively, the results of phenotypic observation, 3,3'-diaminobenzidine(DAB) staining and expression analysis of tobacco immune-related genes revealed that the overexpression of ScLOX1 gene could enhance the defense of N.benthamiana to the F. solani var. coeruleum, but had no significant difference with the control on the defense effect against R.solanacearum. In addition, the expression level of ScLOX1 was down-regulated by methyl jasmine and salicylic acid, but up-regulated by abscisic acid, sodium chloride and polyethylene glycol. The above results provide references for further study on the function of sugarcane ScLOX1 gene.
引文
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[3]张冲.甜瓜脂氧合酶基因家族成员鉴定、表达调控及CmLOX8在果实香气合成中的作用.沈阳农业大学博士学位论文,辽宁沈阳,2016.Zhang C.Identification,Expression and Regulation of Lipoxygenase Gene Family in Melon(Cucumis melo var.makuwa Maldno)and the Role of Cm LOX18 in Synthesis of Fruit Aroma Volatiles.Ph D Dissertation of Shenyang Agricultural University,Shenyang,Liaoning,China,2016(in Chinese with English abstract).
[4]曹嵩晓,张冲,汤雨凡,齐红岩.植物脂氧合酶蛋白特性及其在果实成熟衰老和逆境胁迫中的作用.植物生理学报,2014,50:1096-1108.Cao S X,Zhang C,Tang Y F,Qi H Y.Protein characteristic of the plant lipoxygenase and the function on fruit ripening and senescence and adversity stress.Plant Physiol J,2014,50:1096-1108(in Chinese with English abstract).
[5]Wang R,Shen W B,Liu L L,Jiang L,Liu Y Q,Su N,Wan J M.Anovel lipoxygenase gene from developing rice seeds confers dual position specificity and responds to wounding and insect attack.Plant Mol Biol,2008,66:401-414.
[6]Feussner I,Wasternack C.The lipoxygenase pathway.Annu Rev Plant Biol,2002,53:275-297.
[7]Andreou A,Feussner I,Hause B,Wasternack C,Strack D.Lipoxygenases-structure and reaction mechanism.Phytochemistry,2009,70:1504-1510.
[8]Dubbs W E,Grimes H D.The mid-pericarp cell layer in soybean pod walls is a multicellular compartment enriched in specific lipoxygenase isoforms.Plant Physiol,2000,123:1281-1288.
[9]黄洁雪.水稻种胚脂氧合酶基因Os LOX2的克隆与功能分析.南京农业大学硕士学位论文,江苏南京,2011.Huang J X.Isolation and Characterization of Seed OSLOX2 Gene in Rice.MS Thesis of Nanjing Agricultural University,Jiangsu,Nanjing,China,2011(in Chinese with English abstract).
[10]Axelrod B,Cheesbrough T M,Laakso S.Lipoxygenase from soybeans:EC 1.13.11.12 Linoleate:oxygen oxidoreductase.Method Enzymol,1981,71:441-451.
[11]Melan M A,Dong X N,Endara M E,Davis K R,Ausubel F M,Peterman T K.An Arabidopsis thaliana lipoxygenase gene can be induced by pathogens,abscisic acid,and methyl jasmonate.Plant Physiol,1993,101:441-450.
[12]Ida S,Masaki Y,Morita Y.The isolation of multiple forms and product specificity of rice lipoxygenase.J Agric Chem Soc Jpn,2006,47:637-641.
[13]Ferrie B J,Beaudoin N,Burkhart W,Bowsher C G,Rothstein S J.The cloning of two tomato lipoxygenase genes and their differential expression during fruit ripening.Plant Physiol,1994,106:109-118.
[14]Podolyan A,White J,Jordan B,Winefield C.Identification of the lipoxygenase gene family from Vitis vinifera and biochemical characterisation of two 13-lipoxygenases expressed in grape berries of Sauvignon Blanc.Funct Plant Biol,2010,37:767-784.
[15]Yang X Y,Jiang W J,Yu H J.The expression profiling of the lipoxygenase(LOX)family genes during fruit development,abiotic stress and hormonal treatments in cucumber(Cucumis sativus L.).Int J Mol Sci,2012,13:2481-2500.
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