新疆无苞芥OpNAC083基因的克隆与表达分析

Molecular Cloning and Expression Analyes of OpNAC083 from Olimarabidopsis pumila

本研究通过对新疆耐逆植物无苞芥幼苗c DNA文库的随机克隆测序分析,获得1条与拟南芥NAC转录因子基因At NAC083高度相似的EST序列(Gen Bank登录号为JZ151841),该序列包含1个723 bp最大开放阅读框(ORF),推测编码240个氨基酸。根据该ORF序列设计引物,利用RT-PCR技术从无苞芥叶片的c DNA中克隆了该基因,命名为Op NAC083。比对分析结果表明在其N端具有一段No apical meristem(NAM)保守结构域;该蛋白的二级结构包含33个α-螺旋和11个β-转角。系统进化树分析结果表明,Op NAC083编码产物与拟南芥At NAC083和琴叶拟南芥ANAC083进化关系较近,属于同一进化分支。实时荧光定量PCR(q RT-PCR)分析基因表达结果显示,Op NAC083在无苞芥的不同组织中都有表达,在叶中表达量最高;高盐胁迫处理2 h、ABA处理6 h明显诱导该基因的表达,但PEG-6000和4℃胁迫却抑制该基因的表达。这表明Op NAC083在无苞芥应对盐和ABA胁迫中可能起着重要作用。

Through sequencing of clones randomly selected from previously constructed high quality cDNA library of Olimarabidopsis pumila,we got an expressed sequence tags （EST） which was highly similar to NAC transcription factor gene NAC083 of Arabidopsis thalania.It contained a complete open reading frame （ORF）with 723 bp,designated OpNAC083,encoding 240 amino acids.The primers were designed according to the sequence of ORF and OpNAC083 was identified from O.pumila by RT-PCR and clone techniques.Bioinformatics analysis showed that OpNAC083 contains 33 ct-helixes and l l J3-turns,and a No apical meristem （NAM） at its N-terminal region.Phylogenetic tree analysis revealed that OpNAC083 showed closer genetic kinship with A.thaliana AtNAC083 and A.lyrata ANAC083,indicating that they belonged to the same evolutionary branch. Quantitative Real-Time PCR （qRT-PCR） revealed that OpNAC083 displayed a much broader expression range in O.pumila different tissues,with a maximum expression in leaves.The level of OpNAC083 transcription was upregulated after 2 h of NaCl and 6 h of ABA treatment.However, its transcription was inhibited by PEG-6000 and 4℃ treatment.Our research indicated that OpNAC083 played an important role in response to salt and ABA stress in O.pumila.