青天葵F-box基因的克隆及其与ASK1蛋白互作分析

Cloning of F-box Genes from Nervilia fordii and Analysis of Their Interaction with ASK1 Protein

目的从濒危药用植物青天葵(Nervilia fordii)中克隆F-box基因,为寻找解决青天葵资源紧缺的方法提供依据。方法从青天葵转录组中筛选得到2个F-box基因的核心片段,以RACE技术获得基因的全长并克隆其编码区;通过荧光定量PCR(qRT-PCR)进行组织表达模式分析;利用酵母双杂交实验验证F-box蛋白的F-box区域与拟南芥ASK1蛋白的互作情况。结果从青天葵中克隆到F-box基因NfFBL3和NfFBL4,它们的开放阅读框(open reading frames,ORF)分别长1974、1833 bp,分别编码657、611个氨基酸。二者均包含F-box保守功能结构域,C端具有多个重复亮氨酸序列(Leucine rich repeats,LRRs);NfFBL3和NfFBL4蛋白均为不稳定的疏水蛋白,定位于细胞核,无信号肽及切割位点。系统进化分析表明,NfFBL3和NfFBL4分别与F-box/LRR-repeat protein 3蛋白、F-box/LRR-repeat protein 4蛋白聚在一起。二者均在青天葵的块茎中表达量较高。酵母双杂交实验中NfFBL3的F-box区域能与拟南芥ASK1蛋白互作,而NfFBL4不与ASK1蛋白互作。结论该研究从青天葵中克隆得到2个F-box基因,其中NfFBL3编码的蛋白可与拟南芥ASK1蛋白发生互作,为进一步研究青天葵中Skp1-Cullin-F-box(SCF)复合体的功能提供科学依据。

Objective To clone F-box genes from the endangered medicinal plant Nervilia fordii,and provide a certain scientific basis for finding a solution for the resource shortage of N.fordii.Methods The core fragments of two F-box genes were screened from the transcriptome of N.fordii,the full length of the genes were obtained by RACE technique and their coding regions were cloned;the tissue expression patterns were analyzed by fluorescence quantitative PCR(qRT-PCR).The yeast two-hybrid experiment was used to detect the interaction between the F-box regions domains of the proteins and the Arabidopsis ASK1 protein.Results The F-box genes NfFBL3 and NfFBL4 were cloned from N.fordii.Their open reading frames(ORFs)were 1974 bp and 1833 bp,encoding 657 and 611 amino acids,respectively.NfFBL3 and NfFBL4 contain F-box conserved functional domains and leucine rich repeats(LRRs).They were both unstable hydrophobic proteins,located in the nucleus and none of them had signal peptides or cleavage sites.Phylogenetic analysis showed that NfFBL3 and NfFBL4 clustered with F-box/LRR-repeat protein 3 and F-box/LRR-repeat protein 4,respectively.Both of them expressed highly in the tubers of N.fordii.The F-box domain of NfFBL3 could interact with ASK1 protein from Arabidopsis thaliana,but NfFBL4 could not interact with ASK1 protein in yeast two-hybrid experiment.Conclusion In the current study,two F-box genes were cloned from N.fordii,and the protein encoded by NfFBL3 could interact with ASK1 protein from Arabidopsis thaliana,providing a scientific basis for further study on the function of SKP1-Cullin-F-box(SCF)complex in N.fordii.