甘蔗真核生物翻译起始因子5A基因的克隆与表达分析

Isolation and expression of a eukaryotic translation initiation factor 5A gene from sugarcanefrom sugarcane

真核生物翻译起始因子5A(Eukaryotic translation initiation factor 5A,e IF5A)是一种在动植物和真菌体内普遍存在的蛋白质.为了解e IF5A基因在甘蔗应答生物逆境胁迫中的作用,本研究首先从黑穗病菌胁迫下甘蔗抑制消减杂交(Suppression subtractive hybridization,SSH)文库中获得一条与玉米e IF5A基因(Gen Bank Accession Num ber:EU958725.1)同源性为93%的甘蔗EST序列;其次,以此序列作为探针,通过电子克隆技术获得一条甘蔗e IF5A基因的c DNA拼接序列;最后,经RT-PCR扩增和测序验证,结果显示电子克隆序列与测序序列一致,将该序列命名为Sce IF5A(Gen Bank Acce ssion Number:KJ577595).生物信息学分析显示,Sce IF5A基因c DNA全长1 174 bp,含有长度为483 bp、编码160个氨基酸的完整开放读码框;Sce IF5A蛋白是酸性稳定蛋白,分子量(Mr)为17 453.6,含有12个保守氨基酸序列,推测定位于细胞质.实时荧光定量PCR(Real-time quantitative PCR,RT-qPCR)分析结果表明,在黑穗病菌、水杨酸、茉莉酸甲酯、脱落酸胁迫下,Sce IF5A均上调表达,推测Sce IF5A基因在甘蔗响应黑穗病菌侵染过程中被诱导表达,且其表达受内源激素信号通路的调控.本研究获得的Sce IF5A基因的结构特征和功能作用模式为研究该基因在甘蔗与黑穗病菌互作中的作用积累了基础数据.

Eukaryotic tra nslation initiation factor 5A（e IF5A） is ubiquitous in animals, plants and fungi. To reveal the responses of sugarcane e IF5 A gene to biotic stresses, the present study first obtained an EST sequence with 93% homologs to Zea mays e IF5A（Gen Bank Accession Number：EU958725.1） from a suppression subtractive hybridization（SSH） library of sugarcane challenged by Sporisorium scitamineum. The EST sequence was then used as the probe for in silico cloning to obtain a putative c DNA sequence of sugarcane e IF5 A gene. This gene was validated by reverse transcription-PCR（RT-PCR） amplification sequencing, which showed that the sequence cloned by in silico cloning was in accordance with the sequence amplified by RTPCR method, and was named as Sce IF5A（Gen Bank Accession Number： KJ577595）. Bioinformatics analysis indicated that Sce IF5 A, with a length of 1 174 bp, containing a 483 bp open reading frame（ORF） encoding 160 amino acids protein, was a stable acidic cytopla sm protein with a weight of 17 453.6 Da containing 12 highly conserved amino acid sequences. Realtime quantitative PCR analysis showed that the expression of Sce IF5 A was up-regulated under smut fungus infection and the treatments of SA, Me JA and ABA. The results suggested that Sce IF5 A in sugarcane is most probably involved in response tosuga rcane smut fungus infection and to hormone related bioprocess signaling. The results of this study regarding the structure and function of Sce IF5 A should lay the foundation for further research in the elaboration of functions of this gene during the interaction between sugarcane and S. scitamineum.