小黑麦NAC转录因子基因TwNAC01克隆及生物信息学分析

Cloning and bioinformatics of NAC transcription factor TwNAC01 gene from Triticale(×Triticosecale Wittmack)

NAC(NAM、ATAF1/2、CUC2)是在调控植物发育、衰老、生物与非生物逆境以及激素反应等方面具有重要作用的一类转录因子。为探索六倍体小黑麦耐干旱机制,本研究以六倍体小黑麦为材料,利用快速扩增cDNA末端(RACE)与反转录·聚合酶链反应(RT-PCR)技术克隆到1条NAC转录因子cDNA全长,分析了其生物信息学特点,同时利用实时定量PCR技术分析其在干旱胁迫下的组织部位表达特点。结果表明,所克隆小黑麦基因ORF全长1 059 bp,编码352个氨基酸。该基因预测蛋白中具有NAM亚家族保守的氨基酸序列,且其预测蛋白氨基酸序列与山羊草中的NAC转录因子氨基酸序列同源性高达90%以上。该基因预测蛋白属亲水性蛋白,无跨膜结构域与信号肽区域,二级结构包含45个α-螺旋,57个β-折叠和250个无规则卷曲。预测蛋白定位于细胞核内。在干旱胁迫下,该基因在开花后22 d的小黑麦幼粒和根部的表达量明显高于茎和叶,且表达量显著受干旱胁迫诱导。可见所克隆的基因为小黑麦NAC转录因子,将其命名为TwNAC01(GenBank登录号为MG736919),其在小黑麦抗旱应激反应中具有重要作用。小黑麦NAC转录因子基因TwNAC01的克隆对于揭示小黑麦的抗旱性机制以及小黑麦抗旱基因工程育种具有重要意义。

NAC(NAM、ATAF1/2、CUC2)transcription factors play important roles in regulation of a multitude of traits in plants,such as plant development,senescence,tolerance to biotic and abiotic stresses,and hormone responses.In this study,a novel NAC transcription factor gene from hexaploid triticale was cloned based on RACE and RT-PCR technique,and the gene′s bioinformatics information was analyzed and its gene expression characteristics in different organs under drought stress were determined.The results indicated that the full-length ORF of the gene from hexaploid triticale was 1059 bp,and it was predicted to encode 352 amino acids.The gene predicted protein has the typical conserved domains of NAM subfamily and share more than 90%homology to the NAC protein in Aegilops tauschii.The gene predicted protein is hydrophilic and has no trans-membrane structure,no signal peptide regions.The protein secondary structure contained 45α-helices,57β-sheets and 250 random coils.The predicted protein localizes in the nucleus.The novel gene expresses markly higher in young grains and roots than that in stem or leaves and the gene transcripts were significantly up-regulated under drought stress.These results indicate that the CDS from triticale is a novel NAC transcription factor gene,named as TwNAC 01(Accession number:MG736919 in genebank),which involves in the drought stress resistance response.The cloning of novel NAC transcription factor TwNAC 01 from triticale will help to understand triticale drought resistance mechanism and may hold promise for improving drought stress tolerance in transgenic triticale.