苎麻BnbZIP1转录因子基因的克隆与表达特征分析

doi:10.3864/j.issn.0578-1752.2013.07.002

摘要/Abstract

摘要： 【目的】克隆苎麻转录因子基因BnbZIP1，分析其序列及表达特征，同时进行原核表达以及亚细胞定位分析。【方法】根据苎麻转录组测序中的Unigene48047片段序列，采用RT-PCR结合RACE技术克隆该基因的全长cDNA序列，并进行生物信息学分析；利用Real-time PCR分析该基因在不同组织和不同胁迫条件下表达特征；构建原核表达载体，进行原核表达；构建含EGFP的融合表达载体，观察其亚细胞定位情况。【结果】苎麻BnbZIP1的cDNA全长为2 071 bp，开放读码框为1 407 bp，编码468个氨基酸，推导该多肽的等电点和分子量为7.12和51.57 kD，与毛果杨（XP_002307972）的bZIP基因氨基酸序列的相似性最高，达到93%；IPTG诱导产生的重组蛋白相对分子量约为52 kD，与理论值一致；亚细胞定位分析表明其定位在细胞核中；荧光定量PCR分析表明，该基因在苎麻根、茎、茎尖、叶片、雌花和雄花中均有表达，其中，在雄花中表达最高，在根中表达最低，且该基因受ABA、干旱和高盐诱导上调表达。【结论】获得了BnbZIP1的全长cDNA序列，其编码蛋白具有植物bZIP转录因子典型的结构域，且该基因响应ABA、干旱和高盐逆境胁迫，预示该基因可能在植物抗逆反应中发挥着重要的调控作用。

关键词: 苎麻 , 转录因子 , bZIP , 表达分析 , 亚细胞定位

Abstract: 【Objective】The objective of this study was to clone the full-length cDNA of BnbZIP1 transcription factor gene from ramie, and the expression pattern and the bioinformatics of the sequence were analyzed, and the prokaryotic expression and the subcellular localization were analyzed.【Method】A full-length cDNA sequence was cloned by RT-PCR and RACE methods based on the unigene48047 in ramie transcriptome sequencing. Then the sequence was analyzed through bioinformatics methods and the expression patterns of BnbZIP1 were analyzed by using Real-time PCR in various tissues and under different stress conditions. A prokaryotic expression vector was constructed and the prokaryotic protein expression was induced with IPTG. Then a fusion expression vector containing EGFP was constructed to observe the subcellular localization. 【Result】The full-length cDNA sequence and the ORF of BnbZIP1 were 2 071 bp and 1 407 bp, which encoded 468 amino acids with predicted pI and molecular weight were 4.95 and 36.81 kD, respectively. Homology comparison analysis showed that the deduced BnbZIP1 amino acid sequence shares a 93% homology with bZIP gene (XP_002307972) in Populus trichocarpa. The relative molecular weight of recombinant protein induced by IPTG was 52 kD, which corresponded to the theoretical value. The results of subcellular localization analysis showed that the BnbZIP1 is located in nucleus. The results of real-time PCR suggested that the BnbZIP1 gene expressed in root, stem, shoot tip and blade, female flowers and male flowers, with the highest expression level in male flowers and the lowest in root. The BnbZIP1 gene was up-regulated by ABA, drought and high salt treatment.【Conclusion】The full-length cDNA sequence of BnbZIP1 from ramie was cloned and it had the typical bZIP transcription factor structural domain in plants, and BnbZIP1 gene responses to ABA, drought and high salt stress, which indicated that the BnbZIP1 genes might play an important role in stress response.

Key words: ramie , transcription factor , bZIP , expression analysis , subcellular localization

周精华, 揭雨成, 邢虎成, 钟英丽, 余伟林. 苎麻BnbZIP1转录因子基因的克隆与表达特征分析[J]. 中国农业科学, 2013, 46(7): 1314-1322.

ZHOU Jing-Hua, JIE Yu-Cheng, XING Hu-Cheng, ZHONG Ying-Li, YU Wei-Lin. Cloning and Characterization of the BnbZIP1 Transcription Factor Gene from Ramie (Boehmeria nivea L.)[J]. Scientia Agricultura Sinica, 2013, 46(7): 1314-1322.

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