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[1]孙婷婷 王文举 刘 峰 王 玲 李 竹 戴明剑 阙友雄 许莉萍 苏亚春.甘蔗茉莉酸合成关键酶基因ScOPR1的克隆、亚细胞定位及表达*[J].应用与环境生物学报,2018,24(06):1-14.[doi:10.19675/j.cnki.1006-687x.2018.01005]
 SUN Tingting,WANG Wenju,LIU Feng,et al.Molecular cloning, subcellular localization and expression analysis of jasmonic acid synthesis gene ScOPR1 from sugarcane*[J].Chinese Journal of Applied & Environmental Biology,2018,24(06):1-14.[doi:10.19675/j.cnki.1006-687x.2018.01005]
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甘蔗茉莉酸合成关键酶基因ScOPR1的克隆、亚细胞定位及表达*()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
24卷
期数:
2018年06期
页码:
1-14
栏目:
研究论文
出版日期:
2018-12-25

文章信息/Info

Title:
Molecular cloning, subcellular localization and expression analysis of jasmonic acid synthesis gene ScOPR1 from sugarcane*
文章编号:
201801005
作者:
孙婷婷1 王文举1 刘 峰1 王 玲1 李 竹1 戴明剑1 阙友雄12 许莉萍12 苏亚春12
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 福建福州 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 福建福州 350002
Author(s):
SUN Tingting1 WANG Wenju1 LIU Feng1 WANG Ling1 LI Zhu1 DAI Mingjian1 QUE Youxiong12 XU Liping12 & SU Yachun 12**
1 Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China?
?2 Key Laboratory of Crop Genetics and Breeding and Comprehensive Utilization, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
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
Keywords:
Sugarcane 12-oxo-phytodienoic acid bioinformatic analysis subcellular localization quantitative expression
DOI:
10.19675/j.cnki.1006-687x.2018.01005
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。本研究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cDNA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。该研究结果表明,ScOPR1基因积极响应茉莉酸甲酯、水杨酸信号分子和脱落酸以及黑穗病菌的胁迫。本研究为进一步分析ScOPR1基因的功能提供了参考。图8表1参30
Abstract:
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, none of the report of OPR gene in sugarcane ( Saccharum spp.) was found. In this study, an OPR gene was selected from our established sugarcane transcriptome unigene annotation library. Its full-length cDNA sequence was obtained from ROC22 infected by Sporisorium scitamineum 48 h through 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, a 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 . qRT-PCR analysis revealed that ScOPR1 constitutively expressed in different sugarcane organizations, with the highest expression level in root, followed by in leaf and epidermis, and the minimum amount in bud and stem pith. After methyl jasmonate (MeJA) and salicylic acid (SA) treatments, ScOPR1 gene was significantly up-regulated along with the increase of stress time (3-24 h). At 0.5 h after abscisic acid (ABA) treatment, the expression of ScOPR1 was induced, with 1.54 times higher than that of the control. In addition, ScOPR1 gene was significantly up-regulated at an early infection stage (24 h) in the smut-resistant variety Yacheng05-179 post inoculation with S. scitamineum , but was down-regulated in the smut-susceptible variety ROC22. At 48-72 h, the expression level of ScOPR1 was increased in both two sugarcane genotypes, but its transcripts in the resistant variety were higher than that in the susceptible one. These results demonstrated that ScOPR1 gene was positively response to signal molecules of MeJA, SA and ABA, as well as smut pathogen. The present data has laid a foundation for studying the function of ScOPR1 in the future.

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备注/Memo

备注/Memo:
收稿日期: 2018-01-06 接受日期: 2018-01-29
*福建农林大学杰出青年基金(xjq201630、国家自然科学基金项目(31501363)和现代农业产业技术体系建设专项资金(CARS-17)资助?
**通讯作者(E-mail: syc2009mail@163.com)
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更新日期/Last Update: 2018-04-16