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[1]黄 宁 李聪娜 汤翰臣 郑清雷 凌 辉 陈如凯 阙友雄**.甘蔗泛素结合酶基因的克隆与表达分析[J].应用与环境生物学报,2018,24(04):1-11.[doi:10.3724/SP.J.1145.2017.11006]
 HUANG Ning,ZHENG Qinglei,LI Congna,et al.Cloning and expression analysis of an ubiquitin-conjugating enzyme gene in sugarcane[J].Chinese Journal of Applied & Environmental Biology,2018,24(04):1-11.[doi:10.3724/SP.J.1145.2017.11006]
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甘蔗泛素结合酶基因的克隆与表达分析()
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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04期
页码:
1-11
栏目:
研究论文
出版日期:
2018-08-25

文章信息/Info

Title:
Cloning and expression analysis of an ubiquitin-conjugating enzyme gene in sugarcane
文章编号:
201711006
作者:
黄 宁1 李聪娜1 汤翰臣1 郑清雷1 凌 辉1 陈如凯1 阙友雄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):
HUANG Ning1 ZHENG Qinglei1 LI Congna1 TANG Hanchen1 LING Hui1 CHEN Rukai1 & QUE Youxiong12**
1 Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture, Fujian Agriculture and Forestry University/Sugarcane Research & Development Center, China Agricultural Technology System, 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:
泛素结合酶甘蔗生物信息学实时荧光定量PCR甘蔗黑穗病菌水杨酸茉莉酸甲酯脱落酸
Keywords:
Ubiquitin-conjugating enzymes sugarcane Bioinformatics Real-time quantitative PCR Sporisorium scitamineum SA MeJA ABA
DOI:
10.3724/SP.J.1145.2017.11006
摘要:
目的:克隆鉴定甘蔗泛素结合酶基因(Sugarcane ubiquitin-conjugating enzyme,ScUBc E2 )并探索其在激素信号通路和甘蔗与黑穗病互作过程中的作用。方法:选择黑穗病菌胁迫下甘蔗抑制消减杂交文库(suppression subtractive hybridization, SSH)中注释为泛素结合酶的差异表达EST序列为探针,结合电子克隆技术和RT-PCR技术,以甘蔗cDNA为模板进行泛素结合酶基因克隆。对克隆获得的序列进行生物信息学分析,并利用qRT-PCR技术分析该基因在甘蔗根、蔗髓、叶、芽中的组织特异性表达以及在黑穗病菌、茉莉酸甲酯(Methyl jasmonate,MeJA)、脱落酸(Abscisic acid ,ABA)和水杨酸(Salicylic acid,SA)胁迫下的表达情况。结果:克隆得到一条长度为699 bp的甘蔗泛素结合酶基因(ScUBc E2 , GenBank accession number: KJ599594),该基因包含长度为447 bp的完整开放读码框,编码148个氨基酸。生物信息学分析结果显示,ScUBc E2 编码的蛋白分子量为16.507 kDa;无信号肽,为碱性不稳定的亲水蛋白;包含4个α螺旋,4个β折叠和一些无规则卷曲;第15位氨基酸为泛素化位点,74 ~89位氨基酸为活性位点;在进化过程中与高粱泛素结合酶基因的亲缘关系最近。qRT-PCR分析结果表明,ScUBc E2 基因组成型表达,但在芽中的表达量最高;其表达在甘蔗感黑穗病基因型ROC22中受到黑穗病菌胁迫的抑制,在黑穗病抗病基因型YC05-179中则先被抑制,后被诱导;ScUBc E2 基因受MeJA及SA诱导表达,对ABA胁迫的响应不明显。结论:本研究表明,甘蔗ScUBc E2 基因在抗病基因型和感病基因型甘蔗中存在不同表达模式,可能参与甘蔗与黑穗病菌的互作过程,有望为抗病育种分子标记提供潜在基因资源;同时,该基因受MeJA和SA外源激素胁迫后的表达模式,可为泛素-蛋白酶体途径及激素调控的信号转导在甘蔗与黑穗病菌互作过程中的作用提供一定的理论依据。(图7 表2 参48)
Abstract:
Objective: To study the biological characteristics of sugarcane ubiquitin-conjugating enzyme ( ScUBc E2 ) gene and its role in hormone signaling pathway and in the interaction between sugarcane and Sporisorium scitamineum (S. scitamineum). Methods: An EST that annotated as ubiquitin-conjugating enzyme, was achieved from a sugarcane suppression subtractive hybridization (SSH) library referred to the infection of S. scitamineum. This EST was used as a probe for in silico cloning subsequently, and the cDNA sequence of the sugarcane ubiquitin-conjugating enzyme gene was cloned by RT-PCR and sequenced. The bioinformatics characteristic of this gene was analyzed, and its expression in root, stem pith, leaf and bud, as well as that under the treatment of S. scitamineum, methyl jasmonate (MeJA), abscisic acid (ABA) and salicylic acid (SA) was detected in sugarcane tissue with qRT-PCR method . Results: A 699 bp length of ubiquitin-conjugating enzyme gene ( ScUBc E2 , GenBank accession number: KJ599594) was obtained. The sequence of ScUBc E2 contained a complete open reading frame with a length of 447 bp, which encode 148 amino acids. The bioinformatics analysis showed that the ScUBc E2 was a 16.507 kDa, alkaline unstable hydrophilic protein with no signal peptide. It contain ed 4 alpha helix, 4 be ta folding and some random coil, and its fifteenth amino acid was ubiquitination site and 74th~89th amino acid was active site. Phylogenetic analysis showed that ScUBc E2 had the closest genetic relationship with the homologous gene from sorghum. qRT-PCR analysis indicated that the ScUBc E2 gene was constitutively expressed in sugarcane with the highest level in buds. Besides, the expression of ScUBc E2 gene was inhibited in smut-susceptible genotype ROC22 and in the early smut-infected stage of smut-resistant genotype YC05-179 but was induced at the later smut-infected stage of smut-resistant genotype YC05-179 when the sugarcane plant infected by S. scitamineum. Moreover, the expression of ScUBc E2 gene was also induce by treatment of MeJA and SA in sugarcane tissue culture plantlets, but it was insensitive to ABA. Conclusion: This study showed that the ScUBc E2 gene was involved in the interaction between sugarcane and S. scitamineum. The different expression patterns of ScUBc E2 gene between smut- resistant and -susceptible genotype suggested that ScUBc E2 gene may serve as a molecular marker for disease resistance breeding. At the same time, the expression patterns of ScUBc E2 gene under the stress of MeJA and SA could provide a basis for understanding the role of ubiquitin-proteasome pathway and hormone regulated signal transduction in interaction between sugarcane and S. scitamineum. (Fig ures 9, tables 4 and references 43)

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备注/Memo

备注/Memo:
收稿日期:2017-11-03 接受日期:2017-12-07
*国家自然科学基金(31671752)、福建省杰出青年科学基金(2015J06060) 和国家现代农业产业技术体系(CARS-17)项目资助
**通讯作者(E-mail: queyouxiong@126.com)
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更新日期/Last Update: 2017-12-27