甘蔗茉莉酸合成关键酶基因ScOPR1的克隆、亚细胞定位及表达  

Molecular cloning, subcellular localization, and expression analysis of jasmonic acid synthesis gene ScOPR1 from sugarcane

作　　者：孙婷婷[1] 王文举 刘峰[1] 王玲[1] 李竹[1] 戴明剑 阙友雄[1,2] 许莉萍[1,2] 苏亚春[1,2] SUN Tingting;WANG Wenju;LIU Feng;WANG Ling;LI Zhu;DAI Mingjian;QUE Youxiong;XU Liping;SU Yachun(Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian),Mimstry of Agriculture,Fujian Agriculture and Forestry University,Fuzhou, 350002,China;Key Laboratory of Crop Genetics and Breeding and Comprehensive Utihzation,Ministry of Education,Fujian Agriculture and Forestry University, Fuzhou,350002,China)

机构地区：[1]福建农林大学农业部福建甘蔗生物学与遗传育种重点实验室,福州350002 [2]福建农林大学教育部作物遗传育种与综合利用重点实验室,福州350002

出　　处：《应用与环境生物学报》2018年第6期1365-1374,共10页Chinese Journal of Applied and Environmental Biology

基　　金：福建农林大学杰出青年基金(xjq201630);国家自然科学基金项目(31501363);现代农业产业技术体系建设专项资金(CARS-17)资助~~

摘　　要：12-氧-植物二烯酸还原酶(12-Oxo-phytodienoic acid reductase,OPR)是十八碳烯酸途径的关键酶之一,控制着茉莉酸合成的最后一个步骤.目前,甘蔗(Saccharum spp.)OPR基因的研究尚未见报道.从课题组构建的甘蔗转录组unigene注释库中筛选到一个OPR基因,利用RT-PCR技术从ROC22接种黑穗病菌48 h的蔗芽中扩增获得全长cDNA序列,命名为ScOPR1(GenBank Accession Number:MG755745),并对该基因的序列特征、亚细胞定位、组织特异性表达及其在不同植物激素胁迫和不同基因型甘蔗与黑穗病菌互作过程中的表达情况进行分析.生物信息学分析发现,ScOPR1基因全长1 287 bp,包含1个1 116 bp的开放阅读框,编码371个氨基酸,理论等电点为6.01,含有底物结合活性、FMN结合活性和催化活性的氨基酸保守位点以及OYE家族的保守结构域,且与高粱(Sorghum bicolor)OPR蛋白(XP_002447901.2)的氨基酸序列一致性高达96.23%,推测其为酸性稳定亲水性非分泌OPRⅠ蛋白.本氏烟(Nicotiana benthamiana)瞬时表达显示,ScOPR1::GFP融合蛋白定位于细胞质和细胞膜.qRT-PCR分析结果显示,ScOPR1基因在甘蔗不同组织中均有表达,其在根中的表达量最高,其次为叶和蔗皮,而在蔗芽和蔗肉中的表达量最低.茉莉酸甲酯和水杨酸处理后,随着胁迫时间的延长(3-24 h),ScOPR1基因显著上调表达;该基因在脱落酸处理0.5 h时被诱导表达,为对照的1.54倍.此外,ScOPR1基因在抗黑穗病品种崖城05-179受黑穗病菌侵染初期(24 h)显著上调表达,但在感黑穗病品种ROC22中下调表达;48-72 h时ScOPR1基因的表达量在两个甘蔗基因型中较对照有所增加,但在抗病品种中高于感病品种.本研究表明,ScOPR1基因积极响应茉莉酸甲酯、水杨酸和脱落酸信号分子以及黑穗病菌的胁迫,结果可为进一步分析ScOPR1基因的功能和甘蔗抗病基因工程提供参考.12-Oxo-phytodienoic acid reductase (OPR), one of the key enzymes in the octadecanoid pathway, controls the last step of jasmonic acid synthesis. At present, OPR gene in sugarcane (Saccharum spp.) has not yet been reported. An OPR gene was selected from our established sugarcane transcriptome unigene annotation library. Its full-length cDNA sequence was obtained from ROC22 bud infected with Sporisorium scitamineum for 48 h by using RT-PCR and named as ScOPR1 (GenBank Accession Number: MG755745). Characteristics of ScOPR1 gene sequence, subcellular localization, tissue specific expression, ScOPR1 gene expression level under different plant hormone stresses, and in the interactions of different sugarcane genotypes with smut fungus were analyzed. Bioinformatic analysis showed that the full length of ScOPR1 gene was 1 287 bp, which had an open reading frame of 1 116 bp, encoding 371 amino acids. The pI value of ScOPR1 was 6.01. The conservative amino acids sites, including a catalytic activity, a substrate binding activity, an FMN binding activity and an OYE family structure domain, were discovered in ScOPR1. The similarity of amino acid sequences between ScOPR1 and Sorghum bicolor OPR protein (XP_002447901.2) was 96.23%. Therefore, ScOPR1 was predicted as an acid, stable, hydrophilic, and non-secretory OPRI protein. Transient expression in Nicotiana benthamiana indicated that the ScOPR1::GFP fusion protein was located in the cytoplasm and cell membrane. The qRT-PCR analysis revealed that ScOPR1 was constitutively expressed in different sugarcane organizations, with the highest expression level in root, followed by that in leaf and epidermis, and the minimum amount was noted in bud and stem pith. After methyl jasmonate (MeJA) and salicylic acid (SA) treatments, ScOPR1 gene was significantly up-regulated along with an increase of stress time (3-24 h). At 0.5 h after abscisic acid (ABA) treatment, the expression of ScOPR1 was induced, which was 1.54-fold higher than that of the control. In addition, ScOPR1 gene was sign

关 键 词：甘蔗 12-氧-植物二烯酸还原酶 生物信息学分析 亚细胞定位 定量表达 

分 类 号：Q786[生物学—分子生物学] S566.1[农业科学—作物学]