豆梨NAC转录因子基因PcNAC1的克隆、亚细胞定位及功能初探

Cloning,Sub-cellular Localization and Functional Analysis of the PcNAC1 Gene with NAC Transcription Factor from Pyrus calleryana

根据豆梨(Pyrus calleryana Dence)接种黑斑病菌后的转录组测序结果,筛选出一个上调表达的NAC转录因子基因。从豆梨中获得了该基因的全长片段,并将其命名为PcNAC1。该基因的开放阅读框为792 bp,编码263个氨基酸。将PcNAC1蛋白序列与其他物种蛋白序列进行对比,并构建系统进化树后发现,其与小麦TaNAC4及拟南芥ATAF1具有较高同源性。通过实时荧光定量分析PcNAC1在豆梨不同组中的表达发现,PcNAC1在茎、叶和根中表达量较高,在花和果中表达量较低。构建了亚细胞定位载体,瞬时侵染本氏烟,通过激光共聚焦显微镜观察融合蛋白在细胞内的分布情况,发现该基因行使功能的主要区域定位在细胞核。进一步在本氏烟中瞬时表达PcNAC1,接种烟草疫霉病菌后发现PcNAC1可以通过调控植物激素通路防卫反应基因的表达来增强植物的抗病性。

Up-regulated expression NAC transcription factor was selected from transcriptome sequencing data ofPyrus calleryana Decne infected by Pear black spot. Full length gene was polymerized and entitled as PcNAC1. The open reading frame （ORF） consists on 792 bp nucleotides encoding 263 amino acids polypeptide. Comparison of PcNAC1 with peptide sequences of NAC transcription factors protein family and phylogenetic analysis revealed the highest homology of PcNAC1 with TaNAC4 in Triticum aestivu and ATAF1 in Arabidopsis thaliana. It revealed by Quantitative Real-time PCR that the expression of PcNACl was high in stem, leaf and root of Pyrus calleryana Decne but very low expression in flower and fruit. We synthesized a sub-cellular localization vector to get the transient expression in Nicotiana benthamiana. By confocal laser scanning microscopy it was observed that the fusion protein was localized in nucleus. Furthermore, we infected transgenic Nicotiana benthamiana with Phytophthora parasitica, it was obvious that PcNAC1 enhanced the disease resistance in plant by triggering the expression of pathways of defense response plant hormone genes.