摘要
核糖体循环因子在蛋白质合成体系中发挥着重要作用,利用电子克隆和RT-PCR技术,从花生中克隆到1个核糖体循环因子基因,命名为AhRRF(Genbank登录号KF621303)。序列分析表明,该基因开放阅读框为837 bp,编码278个氨基酸,推测编码蛋白质的分子量为30.94 k D,等电点为9.63,无信号肽序列,AhRRF编码蛋白包含4个α-螺旋、6个β-折叠和一些无规则卷曲结构。系统发生分析结果表明,AhRRF编码蛋白与蒺藜苜蓿、鹰嘴豆、大豆和菜豆的核糖体循环因子具有较高的同源性。实时荧光定量PCR结果显示,AhRRF基因在花生的叶、花、根和幼果中均有表达,但叶中表达量最高,花中最低;UV-B辐射处理1 h时叶中的表达量显著上升,为对照的3.43倍,之后开始下降,24 h时显著低于对照。将构建的重组质粒p ET28a-AhRRF转化大肠杆菌BL21(DE3),以IPTG诱导重组蛋白RRF的表达,SDS-PAGE检测表明,重组蛋白的分子量大小约为30 k D,其大小与推测的大小一致。本研究为进一步探索UV-B辐射对花生分子机理的影响奠定基础。
Ribosome recycling factor（ RRF） plays a important role in the system of protein translation. AhRRF,numbered KF621303,was cloned from Arachis hypogaea L. by the approach of in silico and RT-PCR. sequence analysis showed that the open reading frame of AhRRF was 837 bp. It encodes 492 amino acids with a molecular mass of 30. 94 kD and an isoelectric point of 9. 63,having no signal peptide. The AhRRF protein contains four α- helices,six β-sheets and some random coils. Phylogenetic tree showed that protein AhRRF shares high homology with those from Medicago truncatula,Cicer arietinum,Glycine max,and Phaseolus vulgaris. Real-time quantitative PCR results showed that the expression of gene AhRRF was detected in leaves,flowers,roots and young fruits of A. hypogaea,with the highest expression level in leaves and the lowest in flowers. The expression level AhRRF was significantly increased after UV-B treatment for 1 h,which is 3. 43- folds as high as the control,then it began to drop until significantly below that of the control at 24 h. The recombinant plasmid pET28a-AhRRF was constructed and transformed into E. coli BL21（ DE3）,with the fusion protein His·RRF was expressed by IPTG induction,and SDS-PAGE test showed that His·RRF was about 30 kD,agree with expected size. This study laid the foundation for further research on the molecular mechanisms of peanut affected by UV-B.
出处
《核农学报》
CAS
CSCD
北大核心
2015年第10期1867-1875,共9页
Journal of Nuclear Agricultural Sciences
基金
国家自然科学基金项目(31270461)
浙江省教育厅科研项目(Y201223322)
浙江省植物进化生态学与保护重点实验室开放课题(EEC2014-05)
台州市市级创新团队和重点实验室自主设计科技计划项目(1403KY03)
关键词
花生
核糖体循环因子
克隆
序列分析
原核表达
Arachis hypogaea L.
ribosome recycling factor
gene cloning
sequence analysis
prokaryotic expression
