中间锦鸡儿CiATAF1基因的亚细胞定位及表达分析

Subcellular Localization and Expression Analysis of CiATAF1 Gene in Caragana intermedia

为研究NAC转录因子在非生物胁迫条件下的表达,以中间锦鸡儿为材料,克隆并分析了NAC转录因子家族CiATAF1基因。该基因ORF全长为873bp,编码291个氨基酸,由2个内含子和3个外显子构成,具有典型NAC结构域。染色体步移法获得CiATAF1基因启动子826bp,该启动子含有光、激素、厌氧诱导等多种响应元件。经拟南芥叶片原生质体瞬时表达分析亚细胞定位,CiATAF1基因主要定位于细胞核中。荧光定量分析表明:CiATAF1基因表达具有组织特异性,相比于中间锦鸡儿根和茎,在叶部表达量最高;在干旱、盐、冷、ABA处理下,CiATAF1基因表达量增加,推测该基因可能与非生物胁迫应答相关。CiATAF1同模式植物拟南芥及豆科其他植物的同源基因做多重序列分析,序列一致性为81.14%,表明ATAF1基因序列结构相对保守。生物信息学分析CiATAF1蛋白质属于不稳定的亲水蛋白,二级结构主要由α-螺旋、β折叠、无规则卷曲构成。研究结果对挖掘中间锦鸡儿抗非生物胁迫的基因和深入分析逆境胁迫机理具有重要意义。

To study the expression of NAC under abiotic stress, CiATAF1 gene from Caragana intermedia was cloned and analyzed. It had an ORF of 873 bp and encoded 291 amino acids,consisting of two introns and three exons,and presenting a typical NAC domain. The genome walking method was used to obtain the CiATAF1 promoter with a length of 826 bp,which contained various response elements to light,hormone and anaerobic induction. The subcellular localization was conducted by analysis of the transient expression of Arabidopsis leaf protoplasts,showing that the CiATAF1 gene was mainly located in the nuclei. Quantitative fluorescence RT-PCR analysis showed that the expression of CiATAF1 was tissue-specific ,having the highest expression level in the leaves of Caragana intermedia and relatively low level in roots and stems. Under the treatments of drought,salt,cold and ABA,the expression level of CiATAF1 increased differentially. It was speculated that the CiATAF1 gene is related to the abiotic stress regulations. Multiple sequence analysis was carried out between CiATAF1 with homologues of Arabidopsis and other leguminous plants,and their consistency of the sequences was 81.14%, indicating that the ATAF1 was relatively conservative. Bioinformatics analysis showed that CiATAF1 protein was an unstable hydrophilic protein,and its secondary structure was composed of alpha helixes,beta sheets and random coils. This study had the significance for excavating genes resistant to abiotic stress in Caragana intermedia and for analyzing the mechanism of environmental stresses in depth.