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甘蔗脂氧合酶基因ScLOX1的克隆与表达分析 预览

Cloning and expression analysis of sugarcane lipoxygenase gene ScLOX1
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摘要 LOX属于脂氧合酶超家族(lipoxygenase superfamily),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PLATLH2和Lipoxygenase活性位点,与高粱(Sorghum bicolor) LOX (XP002466613.1)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 cDNA 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 ScLOX1 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-traditional 9-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 ja
作者 孙婷婷 王文举 娄文月 刘峰 张旭 王玲 陈玉凤 阙友雄 许莉萍 李大妹 苏亚春 SUN Ting-Ting;WANG Wen-Ju;LOU Wen-Yue;LIU Feng;ZHANG Xu;WANG Ling;CHEN Yu-Feng;QUE You-Xiong;XU Li-Ping;LI Da-Mei;SU Ya-Chun(Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture / Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China;Key Laboratory of Crop Genetics and Breeding and Comprehensive Utilization, Ministry of Education / Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China)
出处 《作物学报》 CAS CSCD 北大核心 2019年第7期1002-1016,共15页 Acta Agronomica Sinica
基金 国家自然科学基金项目(31501363) 福建省高校杰出青年科研人才计划项目(苏亚春-2017) 福建农林大学杰出青年基金(xjq201630) 国家现代农业产业技术体系建设专项(CARS-17)资助.
关键词 甘蔗 脂氧合酶 生物信息学 实时荧光定量PCR 生物和非生物胁迫 sugarcane lipoxygenase bioinformatics real-time flourescent quantitative PCR biotic and abiotic stresses
作者简介 第一作者联系方式:孙婷婷,E-mail:sunting3221@163.com;通信作者:苏亚春,E-mail:syc2009mail@163.com.
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