胡杨NAC转录因子PeNAC045基因的克隆及功能分析

Cloning and functional analysis of Pe NAC045 from Populus euphratica

NAC(NAM、ATAF1/2和CUC2)域蛋白是植物特有的最大的转录因子家族之一,在调节衰老,细胞分裂,木质形成,生物和非生物胁迫等方面发挥重要作用。本研究从胡杨中成功克隆出与胁迫相关的基因,并命名为PeNAC045。测序结果表明,PeNAC045基因编码区长度为915 bp,编码304个氨基酸,与毛果杨PtrNAC045的氨基酸一致性为96.05%。对PeNAC045基因在NaCl和干旱胁迫下的表达情况进行了分析,PeNAC045基因的表达受高盐和干旱的强烈诱导。利用PeNAC045的cDNA全长构建表达载体pBI121-PeNAC045-GFP,测序确认后,将重组结构和阳性对照(空载体)分别转化野生型拟南芥(Col-0),进行亚细胞定位观察,结果显示PeNAC045-GFP融合蛋白定位于细胞核上,并由DAPI进行核染色标定。利用农杆菌花序侵染法将构建的表达载体pCAMBIA1301-Pe NAC045转化野生型拟南芥和突变体(ataf2),通过PCR鉴定,获得过表达植株及ataf2/PeNAC045回补株系。对各株系进行NaCl胁迫处理,分析PeNAC045基因的生物学功能。在150 mmol/L NaCl胁迫下,相比于拟南芥突变体和野生型植株,拟南芥PeNAC045过表达株系的萌发率降低,根长变短。此外,拟南芥PeNAC045过表达株系的株高明显低于其他株系,其在苗期对盐胁迫的敏感性增加。研究结果表明,在盐胁迫下,PeNAC045作为转录调节因子,负调控胁迫相关基因的表达。

NAC （NAM, ATAF1/2 and CUC2） domain proteins constitute one of the largest plant-specific transcription factors （TFs） and play an important role in regulating senescence, cell division, wood formation and biotic and abiotic stresses. In present study, we successfully isolated a stress responsive gene from Populus euphratica, i. e. , PeNAC045. Sequencing results indicated that the length of PeNAC045 is 915 bp encoding 304 amino acids, and PeNAC045 shares 96.05% homology in amino acid sequence with PtrNAC045. The expression of PeNAC045 in response to NaCl and drought stress was characterized. PeNAC045 mRNA expression was strongly induced by high-salinity and drought treatment.The expression vector pBI121-PeNAC045-GFP was constructed using the full-length PeNAC045 cDNA cloned into the pBI121-GFP vector. After sequencing confirmation, the construct and positive control （empty vector） were transformed into Arabidopsis. Subcellular localization experiments in Arabidopsis indicated that the PeNAC045-GFP fusion protein was localized in the nucleus. Arabidopsis was stained with the DNA dye 4,6-diamidino-2-phenylindole （DAPI） to visualize the nucleus. The expression vector pCAMBIA1301-PeNAC045 was constructed and transformed into Arabidopsis thaliana wild type （Col-0） and ataf2 mutant using floral dip method. Then, we obtained the PeNAC045 overexpression lines of Arabidopsis and ataf2/PeNAC045. To test the function of PeNAC045, each line was treated with NaCl.The germination percentage of PeNAC045 overexpression lines of Arabidopsis was reduced and the root length was shorter under 150 mmol/L NaCl stress, compared to ataf2 mutant and wild-type plants.Furthermore, the sensitivity of transgenic PeNAC045 overexpression lines to NaCl stress was increased at the seedling stage and the height of the seedlings was significantly lower than others. Our results indicated that PeNAC045, as a transcriptional regulator, negatively regulates the expression of stress responsive genes under NaCl stress.