杨树PeAFB基因克隆及表达模式初步分析

doi:10.11707/j.1001-7488.20150804

林业科学 ›› 2015, Vol. 51 ›› Issue (8): 26-32.doi: 10.11707/j.1001-7488.20150804

杨树PeAFB基因克隆及表达模式初步分析

陈英, 邵志龙, 王浩然, 朱燕宇, 朱嵊, 黄敏仁

南京林业大学南方现代林业协同创新中心江苏省农业种质资源保护与利用平台杨树种质资源圃南京 210037

收稿日期:2014-10-09 修回日期:2015-01-13 出版日期:2015-08-25 发布日期:2015-09-10
通讯作者: 黄敏仁
基金资助:
林业公益性行业科研专项(201304111); 国家自然科学基金项目(31200507); 江苏省基础研究计划(自然科学基金)-面上项目研究(BK2012817); 国家自然科学基金项目(31170620); 江苏省高校优势学科建设工程资助项目(PAPD)。

Cloning and Expression of PeAFB Genes in Populus

Chen Ying, Shao Zhilong, Wang Haoran, Zhu Yanyu, Zhu Sheng, Huang Minren

Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University Poplar Germplasm Nursery, The Jiangsu Provincial Platform for Conservation and Utilizations for Agricultural Germplasm Nanjing 210037

Received:2014-10-09 Revised:2015-01-13 Online:2015-08-25 Published:2015-09-10

摘要/Abstract

摘要：

[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2 461,3 115,2 441,2 818 bp,对应的ORF分别为1 546,1 716,1 716,1 914 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(500 μmol·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。

关键词: NL-895杨, TIR1/AFB F-box蛋白, PeAFB, 胁迫应答

Abstract:

[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 PeAFB 5 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.

Key words: poplar NL-895, TIR1/AFB F-box protein, PeAFB, stress responses

中图分类号:

S718.46

陈英, 邵志龙, 王浩然, 朱燕宇, 朱嵊, 黄敏仁. 杨树PeAFB基因克隆及表达模式初步分析[J]. 林业科学, 2015, 51(8): 26-32.

Chen Ying, Shao Zhilong, Wang Haoran, Zhu Yanyu, Zhu Sheng, Huang Minren. Cloning and Expression of PeAFB Genes in Populus[J]. Scientia Silvae Sinicae, 2015, 51(8): 26-32.

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杨树PeAFB基因克隆及表达模式初步分析

Cloning and Expression of PeAFB Genes in Populus

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