无苞芥NAC转录因子基因OpNAC026的克隆与分析

Clone and Expression of NAC Transcription Factor Gene OpNAC026 from Olimarabidopsis pumila

在新疆耐逆植物无苞芥幼苗cDNA文库的随机克隆测序结果中,发现1条与拟南芥NAC转录因子基因AtNAC026高度相似的5′端EST序列(GenBank登录号为JZ151854),对该克隆进行3′端测序,拼接得到一条全长1 327bp的cDNA序列,该序列包含一个906bp的最大开放阅读框(ORF),推测编码301个氨基酸。根据该ORF序列设计引物,利用RT-PCR技术对其进行克隆,将该基因命名为OpNAC026(GenBank登录号为KM457621)。理化性质分析表明,OpNAC026蛋白是一个无跨膜区域的亲水蛋白,在N端具有一段保守结构域;蛋白质二级结构预测显示,OpNAC026蛋白包含54个α-螺旋和12个β-转角。系统进化树分析表明,OpNAC026基因与拟南芥AtNAC026和AtNAM进化关系最近。实时荧光定量PCR(qRT-PCR)分析显示,OpNAC026基因在无苞芥的各组织中都有表达,在叶中表达量最高;高盐胁迫处理24h、干旱12h、ABA 6h、4℃低温8h处理均可明显诱导OpNAC026基因的表达。研究表明,OpNAC026基因可能参与无苞芥抗逆机制的调控。

An expressed sequence tag（GenBank accession number：JZ151854） from the 5＇ end which was highly similar to NAC transcription factor AtNAC026 of Arabidopsis thalania was obtained, through sequencing of clones randomly selected from the previously constructed cDNA library of Olimarabidopsis pumila leaves. This clone was sequenced from the 3＇ end,and a full-length cDNA of 1 327 bp was obtained after sequence assembly. Its open reading frame （ORF） was 906 bp,and encodes 301 amino acids. The primers were designed according to the ORF and this gene was cloned from O. pumila by RT-PCR technique,which was designated as OpNAC026 （GenBank accession number： KM457621）. Physicochemical property analysis showed that OpNAC026 is a hydrophilic protein with a conserved domain at its N-termi- nal region,and OpNAC026 has no transmembrane domain,indicating that it is not a transmembrane protein. The secondary structural analysis showed that OpNAC026 contains 54 a-helixes and 12 13-turns. The phylogenetic analysis revealed that OpNAC026 showed the closest genetic kinship with A. thaliana At- NAC026 and AtNAM, indicating that they belonged to the same evolutionary branch. Quantitative realtime PCR （qRT-PCR） revealed that OpNAC026 displayed a much broader expression range at O. pumila different tissues, with a maximum expression in leaves. The transcriptional level of OpNAC026 was up-regulated as 24 h of NaCl treatment,12 h of 20% PEG-6000 treatment,6 h of ABA treatment and 8 h of 4 ℃ treatment,respectively. Our research indicated that OpNAC026 might control the mechanism of resistance in O. pumila.