日本结缕草ZjNAC2基因的克隆、亚细胞定位及表达分析

Molecular cloning, subcellular localization analysis, and expression characterization of ZjNAC2 from Zoysia japonica

NAC(NAM,ATAF1/2,CUC2)是植物所特有的最大的转录因子家族之一,在调控植物生长发育、响应非生物胁迫等方面发挥着重要作用。本研究利用cDNA末端快速扩增技术(rapid amplification of cDNAends,RACE)从日本结缕草(Zoysiajaponica)中克隆获得了ZjNAC2基因(GenBank登录号为MH580281),其编码区序列长1110bp,编码369个氨基酸。保守结构域分析显示,ZjNAC2编码蛋白在N端有一个典型的NAM结构域,表明ZjNAC2属于NAC转录因子家族。同时通过染色体步移的方法获得其启动子序列1574bp,通过作用元件分析发现该启动子上有ABA、非生物胁迫等多个不同的响应元件。亚细胞定位结果显示,ZjNAC2定位于细胞核。荧光定量分析表明,ZjNAC2在根中的表达量最高,并且在衰老叶片中表达量显著高于幼嫩叶片;此外ZjNAC2的表达受200μmol·L^-1ET、10μmol·L^-1MeJA、10μmol·L^-1ABA和20%PEG4000的抑制,但受300mmol·L^-1NaCl处理的诱导。本研究表明ZjNAC2转录因子可能参与多种信号转导途径,这为进一步深入研究ZjNAC2的功能奠定了基础。

NAC is one of the largest transcription factor families unique to plants,and plays an important role in regulating plant growth and development and responding to abiotic stress.In this study,the ZjNAC2 gene was cloned from Zoysia japonica using the RACE method.Its coding region is 1 110 bp in length,encoding 369 amino acids.Conserved domain analysis showed that the ZjNAC2 has a typical NAM domain at the N-terminus,indicating that ZjNAC2 belongs to the NAC transcription family.At the same time,the 1 574 bp promoter sequence was obtained using a genome walking method.Ciselements prediction demonstrated that there were many different response elements within the promoter,such as ABA and abiotic stress-responsive cis-elements.Subcellular localization results showed that ZjNAC2 was located in the nucleus.Realtime quantitative PCR analysis showed that ZjNAC2 was expressed most abundantly in roots and was significantly enriched in senescent leaves.In addition,ZjNAC2 expression was inhibited by 200 μmol·L^-1 ET,10 μmol·L^-1 MeJA,10 μmol·L^-1 ABA,or 20% PEG4000,but induced by 300 mmol·L^-1 NaCl treatment.This study demonstrates that ZjNAC2 is an important transcription factor which could be involved in multiple signaling pathways.The present study paves the way to further study the functions of ZjNAC2 in Zoysia japonica.