草果AtNCED基因克隆、表达和植物表达载体构建

9-顺式环氧类胡萝卜素双加氧酶(9-cis-epoxycarotenoid dioxygenase, NCED)是ABA生物合成的限速酶。诸多研究表明NCED基因表达量的变化与ABA含量显著相关,在种子休眠过程中发挥重要作用。为探究草果AtNCED基因的序列特征和功能,本研究以草果(Amomum tsaoko Crevost et Lemarie)种子转录组为基础,采用RT-PCR技术克隆了一个AtNCED基因。该基因全长2 372 bp,包含一个1 830 bp的开放阅读框(open reading frame, ORF),编码610个氨基酸。生物信息学分析显示,AtNCED基因编码的蛋白在N-末端包含典型的叶绿体转运肽序列,在催化结构域含有4个高度保守的组氨酸残基。AtNCED蛋白与芭蕉科马来西亚蕉同源性较高,与其他单子叶植物处在同一进化分支。实时荧光定量PCR分析结果表明,AtNCED基因表达量随着层积时间的增加逐渐下降,该基因可能正向调控种子休眠,在草果种子休眠诱导与维持中发挥重要作用。进一步地,利用同源重组方法,成功构建了pBI121-AtNCED过表达载体,并转化至农杆菌EHA105。该研究结果为弄清ABA激素信号在种子休眠中的调控作用提供了帮助。

Cloning,Expression and Plant Expression Vector Construction of AtNCED Gene from Amomum tsaoko

9-cis-epoxycarotenoid dioxygenase(NCED) is considered to be the rate-limiting enzyme and play a pivotal role in ABA biosynthesis. Many studies have demonstrated that the gene expression of NCED was significantly associated with ABA level, and played an important role in the process of seed dormancy. Aimed to study the characteristics and functions of NCED, we cloned a NCED gene(designated as AtNCED) from Amomum tsaoko on the basis of the seed transcriptome data through the RT-PCR technique. Sequence analysis showed that the full length cDNA of AtNCED was 2 372 bp and contained a complete open reading frame(ORF) of 1 830 bp, which encoded 610 amino acid residues. Bioinformatic analysis showed that AtNCED protein contained a putative chloroplast targeting peptide at N-terminus region, and 4 highly conserved histidine residues in the catalytic domain. AtNCED protein showed high homology to Ma NCED protein, and it was at the same evolutionary branch with other monocotyledon. Quantitative real-time PCR analysis showed that the gene expression of AtNCED decreased gradually with stratification time increased, AtNCED gene may play a positive role in the regulation of seed dormancy and control the induction and maintenance of seed dormancy. Furthermore, we successfully constructed overexpression vector pBI121-AtNCED by homologous recombination method and transformed it to Agrobacterium tumefaciens stain EHA105, which provided a foundation work for further revealing the role of ABA signal in seed dormancy of A. tsaoko.