摘要
为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,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,Me JA）、脱落酸（Abscisic acid,ABA）和水杨酸（Salicylic acid,SA）胁迫下的表达情况.最终克隆得到一条长度为699 bp的甘蔗泛素结合酶基因（ScUBc E2;Gen Bank accession number：KJ577594.1）,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（Mr）为16.507×10^3;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.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.
The objective of this study was to examine the biological characteristics of the sugarcane ubiquitin-conjugating enzyme（Sc UBc E2） gene, its role in the hormone signaling pathway, and the interaction between sugarcane and Sporisorium scitamineum. An EST was annotated as the ubiquitin-conjugating enzyme from a sugarcane suppression subtractive hybridization（SSH） library from the infection of S. scitamineum. This EST was subsequently used as a probe for in silicocloning, and the c DNA sequence of the sugarcane ubiquitin-conjugating enzyme gene was cloned using RT-PCR and was then sequenced. The bioinformatic characteristics of this gene was analyzed, and q RT-PCR was used to detect expression in sugarcane tissues, including the root, stem pith, leaf, and bud, and when treated with S. scitamineum, methyl jasmonate（Me JA）, abscisic acid（ABA）, and salicylic acid（SA）. Finally, a ubiquitin-conjugating enzyme gene（Sc UBc E2, Gen Bank accession number： KJ577594.1） that was 699 bp was obtained. The sequence of Sc UBc E2 contained a complete open reading frame of 447 bp that encodes 148 amino acids. The bioinformatics analysis showed that Sc UBc E2 was 16.507 × 10^3 and an alkaline unstable hydrophilic protein with no signal peptide. It contained 4 alpha helix, 4 beta folding, and some random coils,its fifteenth amino acid was a ubiquitination site, and the 74 th-89 th amino acid was an active site. The phylogenetic analysis showed that ScUBc E2 had the closest genetic relationship to a homologous gene in sorghum. The qRT-PCR analysis indicated that the ScUBc E2 gene was constitutively expressed in sugarcane with the highest level in the buds. The expression of the ScUBc E2 gene was inhibited in the smut-susceptible genotype ROC22 and in the early smut-infected stage of the smut-resistant genotype YC05-179, but it was induced at the later smut-infected stage in the smut-resistant genotype YC05-179 when the sugarcane plant was infected by S. scitamineum. Moreover, the expression of the ScUBc E2 gene was induced by the treatment of MeJA and SA in the sugarcane tissue culture plantlets, but it was insensitive to ABA. This study showed that the ScUBc E2 gene was involved in the interaction of sugarcane and S. scitamineum. The different expression patterns of the ScUBc E2 gene and the smut-resistant and smut-susceptible genotypes suggest that the ScUBc E2 gene may serve as a molecular marker for disease resistance during breeding. The expression patterns of the ScUBc E2 gene under the stress of MeJA and SA could also provide a basis for understanding the role of the ubiquitin-proteasome pathway and hormone regulated signal transduction in the interaction of sugarcane and S. scitamineum.
作者
黄宁
李聪娜
汤翰臣
郑清雷
凌辉
陈如凯
阙友雄
HUANG Ning;LI Congna;TANG Hanchen;ZHENG Qinglei;LING Hui;CHEN Rukai;QUE Youxiong(Ministry of Agriculture Key Laboratory of Sugarcane Biology and Genetic Breeding（Fujian）,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Ministry of Education Key Laboratory of Crop Genetics and Breeding and Comprehensive Utilization,Fujian Agriculture and Forestry University,Fuzhou 350002,China)
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2018年第4期845-852,共8页
Chinese Journal of Applied and Environmental Biology
基金
国家自然科学基金项目(31671752)
福建省杰出青年科学基金项目(2015J06060)
国家现代农业产业技术体系(CARS-17)项目资助~~
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
ubiquitin-conjugating enzyme
sugarcane
bioinformatics
real-time quantitative PCR
Sporisorium scitamineum
salicylic acid （SA）
methyl jasmonate （MeJA）
abscisic acid （ABA）
