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期 刊 ：植物遗传资源学报 2014年 15卷 1期

关键词：苹果砧木；MdNCED1；克隆；表达；原核表达；

Keywords:apple rootstocks, MdNCED1, cloning, expression, prokaryotic expression,

摘 要 ：以苹果砧木SH40（Malus domestica ?Malus Honanensis）为试材，采用RT-PCR结合RACE技术从苹果茎尖中克隆得到一个脱落酸（ABA）代谢合成关键基因：9-顺式-环氧类胡萝卜素双加氧酶（NCED）基因cDNA序列全长，命名为MdNCED1，其全长2179bp,编码606个氨基酸。该基因在GenBank登录号为KC816734。氨基酸同源性分析表明，MdNCED1与已报道的其他植物物种的氨基酸序列具有63.7%-93.0%的相似性。通过构建MdNCED1基因的原核表达载体pDEST15-MdNCED1，转入大肠杆菌（DE3），用IPTG诱导。SDS-PAGE分析表明，MdNCED1基因在大肠杆菌中被诱导表达蛋白质分子量与预期结果一致。荧光定量结果表明，MdNCED1基因在SH28、M26、SH40及其嫁接品种“嘎啦”的表达趋势均呈先上升后下降的过程，同时与其矮生程度呈正相关。

Abstract:9-cis-epoxycarotenoid (NCED) gene is a critical gene in the pathway of abscisic acid biosynthesis. In this research, NCED gene in apple, designated as MdNCED1 (GenBank accession number: KC816734), was isolated from the apical tissue of apple rootstock SH40 (M. domestica ?M. Honanensis）by RT-PCR and RACE technology. The full cDNA was 2179bp，containing a complete open reading frame that encodes 606 amino acids. Amino acid sequence analysis revealed that the sequence had 63.7%-93.0% similarity with those of other reported plants. The ORF fragment of the gene was cloned into vector pDEST15，the recombined plasmid was transformed in to E. coli. (DE3). The result of SDS-PAGE demonstrated that the size of expected protein in the prokaryotic expression system. Fluorescent quantitative PCR analysis showed that the trend of expression of SH28, M26, SH40 and the grafted varieties ‘Gala’ were increased and then decreased. And the expression of this gene was positively associated to the dwarfing degree.

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