杨树PeAFB基因克隆及表达模式初步分析

摘要

Objective]The TIR1/AFB F-box proteins are auxin receptors,which directly involve in auxin perception by degrading Aux/IAA proteins,thereby regulating biological processes of stress responses and development. In order to reveal the auxin signal transduction and regulation mechanisms of AFB genes in poplar,AFB gene family members of poplar were cloned and their expression patterns under non-host pathogen,plant hormone and drought treatment were analyzed.[Method]Using PCR and RACE methods,AFB gene family members were cloned from a poplar hybrid clone NL-895 ( Populus deltoides × P. euramericana cl. NL-895 ) . Online softwares and databases were used for gene predictions,conserve domain prediction,tertiary structure prediction,multiple-sequence alignments,and phylogenetic analysis. Real-time RT-PCR was conducted for gene expression pattern analysis of poplar AFB genes.[Result]Four AFB gene family members in NL-895 were cloned and named PeAFB2-1,PeAFB2-2,PeAFB3 and PeAFB5 respectively. The full-length cDNA sequences of the PeAFB genes were 2 461 ,3 115 ,2 441 ,and 2 818 bp and the corresponding lengths of ORF (open reading frames) were 1 546,1 716,1 716 and 1 914 bp. AFBs are usually leucine-rich proteins,and the 4 PeAFBs also show high leucine content ( 10% -13. 4%) . The analysis of deduced protein sequences showed that 4 PeAFBs all contained a typical conserved F-box domain ( IPR001810 ) and five LRR-rich repeats ( IPR006553 ) . A phylogenetic analysis using the 47 AFB proteins from land plants revealed that all AFB proteins fell into four clades,and the 4 PeAFBs resided in the first clade. Furthermore, four PeAFB genes’ expressions were down-regulated under Pseudomonas syringae DC3000,MeJA(500 μmol·L -1),and PEG(20%,m/V) treatment.[Conclusion]Four PeAFB gene family members in poplar NL-895 were cloned,and their phylogenetic relationships were revealed. Four PeAFB genes’expressions were down-regulated under biotic and abiotic stresses,which indicate the four genes may play a role in negative regulation of poplar abiotic/biotic stress responses.%【目的】TIR1/AFB F-box作为生长素受体参与从生长素结合到 Aux/IAA蛋白降解这一信号转导过程。通过克隆杨树 AFB基因家族成员，并检测其在病原菌、激素和干旱胁迫下的表达模式，以期了解 AFB 基因在杨树生长发育及激素信号转导中的作用机制。【方法】以美洲黑杨杂种 NL-895杨为材料，通过 PCR 与 RACE 技术，克隆 NL-895杨 AFB基因家族成员全长 cDNA，进而利用在线分析软件和数据库对各成员编码蛋白质的理化参数、保守结构域、蛋白质三级结构进行分析和预测；根据氨基酸序列多重比对结果进行系统进化分析；采用实时定量PCR技术研究4个NL-895杨AFB基因家族成员在病原菌、激素和干旱胁迫下的表达模式。【结果】获得了4个 NL-895杨 AFB基因家族成员，分别命名为 PeAFB2-1，PeAFB2-2，PeAFB3和 PeAFB5，其全长 cDNA分别为2461，3115，2441，2818 bp，对应的 ORF分别为1546，1716，1716，1914 bp。4个 PeAFB蛋白亮氨酸含量均在10%以上，特别是 PeAFB2-1，高达13.4%，符合 AFB蛋白质富含亮氨酸特征。氨基酸序列分析结果表明，PeAFB家族成员均具有 F-box蛋白特征，其 N－端为典型的 F-box 结构域( IPR001810)，还含有5个富含亮氨酸重复序列(LRR-rich repeat，IPR006553)。4个杨树 AFB和其他模式植物 TIR1/AFB F-box 蛋白所构建系统发育树将47个 AFB蛋白家族成员聚为4大类，4个杨树 PeAFB蛋白属于第Ⅰ类，并且与拟南芥的 AtAFB 蛋白家族成员具有高度相似性。4个 PeAFB基因在茉莉酸甲酯(500μmol·L －1)、丁香假单胞菌 DC3000和 PEG(20%，m/V)等胁迫条件下，表达量均有不同程度的下调。【结论】本研究克隆了4个杨树 PeAFB家族成员，并揭示了其系统进化关系，基因表达分析表明4个 PeAFB基因在生物和非生物胁迫条件下，其表达量均有不同程度的下调，表明其可能参与了杨树对胁迫应答的负调控。